Novel combination of anticholinergics-b2-adrenoceptor agonists, antileukotrienes (leukotriene receptor antagonists), glucocorticoids and/or phosphodiesterase 4 inhibitors for the treatment of inflammatory diseases

ABSTRACT

The invention relates to novel combinations based on anticholinergics, β 2 -adrenoceptor agonists, PDE 4 Inhibitors, glucocorticoids, and leukotriene-receptor antagonists, process for their production and their use for the treatment of inflammatory diseases, preferably respiratory diseases as bronchial asthma and chronic obstructive pulmonary diseases (COPD) or rheumatic or autoimmune diseases.

This application is a divisional of U.S. patent application Ser. No.11/642,967, filed Dec. 21, 2006, which claims the benefit of U.S.Provisional Application No. 60/752,058 filed on Dec. 21, 2005, all ofwhich are incorporated herein by reference in their entirety.

The invention relates to novel combinations based on anticholinergics,β₂-adrenoceptor agonists, PDE 4 Inhibitors, glucocorticoids, andleukotriene-receptor antagonists, process for their production and theiruse for the treatment of inflammatory diseases, preferably respiratorydiseases as bronchial asthma and chronic obstructive pulmonary diseases(COPD) or rheumatic or autoimmune diseases.

Asthma bronchiale, affecting as many as 10% of individuals inindustrialized nations, is characterized by bronchoconstriction, chronicairway inflammation, airway hyperreactivity, and mucosal oedema. Airwayremodelling and altered noncholinergic, nonadrenergic neurotransmissionmay contribute to irreversible airway obstruction and reduction ofpulmonary function. Asthma is characterized by recurrent episodes ofbreathlessness, wheezing, coughing, and chest tightness, termedexacerbations. The severity of exacerbations can range from mild to lifethreatening. The exacerbations can be a result of exposure to e.g.respiratory infections, dust, mould, pollen, cold air, exercise, stress,tobacco smoke, and air pollutants. Bronchial asthma has emerged as amajor public health problem worldwide over the past 20 years. Althoughdata indicate that current asthma therapies led to limited decreases indeath rates, it continues to be a significant health care problem. Itstill is one of the leading causes of preventable hospitalizationworldwide and accounts for several million lost workdays. Along with theincrease in asthma prevalence, the costs associated with this diseasehave also risen dramatically.

Chronic obstructive pulmonary disease (COPD) is very common world-wide.It is primarily associated with past and present cigarette smoking butthe fact that the society gets older also plays an even continuouslyincreasing role. The prevalence of COPD varies between 3% and 10% with asteadily increasing trend. Although COPD is a leading cause of illnessand death, its recognition as a public health problem has been slow toevolve despite the rising mortality rate for COPD and the decline indeath rates for most of the cardiovascular diseases (Hurd Chest 2000;117(2 Suppl):1S-4S). Additionally, COPD imparts substantial economicburden on individuals and society. COPD is a disease characterised bychronic inflammation and irreversible airflow obstruction with a declinein the lung function parameter FEV₁ that is more rapid than normal. Thedisease has two major aspects of pathology, namely chronic bronchitis,characterised by mucus hypersecretion from the conducting airways, andemphysema, characterised by destructive changes in the alveoli.

Asthma and COPD are serious world health problems characterised by achronic inflammatory disorder of the airways. Airflow obstruction andairway inflammation are features of asthma as well as COPD. Although theairway inflammation in asthma and COPD, respectively, involve differentcell types, both diseases are of chronic inflammatory nature associatedwith cellular infiltration and activation. While bronchial asthma ispredominantly characterized by eosinophils and CD4+ lymphocytes,neutrophil granulocytes, CD8+ lymphocytes and macrophages appear to playa major role in the pathogenesis of COPD (Saetta et al. Am J Respir CritCare Med 1999; 160:711-7, Shapiro Am J Respir Crit Care Med 1999;160:S29-S32).

There is now strong evidence that airway inflammation is a predominantunderlying problem in patients with asthma and COPD.

The pathophysiology of asthma involves an interactive network ofmolecular and cellular interactions, although the contribution of eachindividual factor is probably different from patient to patientdepending on the setting and stimulus. Major participants in thedevelopment of an asthma phenotype include the triggering stimuli suchas the allergens themselves, cells such as T cells, epithelial cells andmast cells that produce a variety of cytokines including tumor necrosisfactor-alpha (TNFα), interleukin (IL)-5, granuloyte-macrophagecolony-stimulating-factor (GM-CSF), IL-3, IL-4 and IL-13 and chemokinessuch as eotaxin, adhesion molecules, etc.

Unfortunately, much less is known about the pathogenesis of COPD thanthat of asthma. Recent studies have greatly expanded the understandingof pathogenetic mechanisms underlying COPD. Thus, there is consent thatCOPD is also an inflammatory disease. From the present pathogeneticalpoint of view, COPD is defined as a progressive and not fully reversibleairway obstruction with predominantly neutrophilic airway inflammation.In COPD, the predominant inflammatory cell types are CD8⁺ T lymphocytes,macrophages, and neutrophils. Neutrophils and neutrophilic inflammatorymarkers including IL-8, TNFα and LTB₄ are increased in the airways ofCOPD patients (Yamamoto et al. Chest 1997; 112:505-10, Keatings et al.Am J Respir Crit Care Med 1996; 153:530-4).

The current management of asthma and COPD is focussed on the improvementof the lung function of patients. In both diseases but especially inCOPD, the first step is smoking cessation. There is evidence thatsmoking reduction or cessation may result in improvement of somerespiratory parameters.

Bronchoconstriction occurs due to bronchial smooth muscle spasm andairway inflammation with oedema. β₂-adrenoceptor agonists provide abronchodilator effect to the patients, resulting in relief from thesymptoms of breathlessness. β₂-adrenoceptor agonists can be short-actingfor immediate relief, or long-acting for long-term prevention ofrespiratory symptoms. Short-acting β₂-adrenoceptor agonists currentlyavailable include salbutamol, terbutaline, reproterol, pirbuterol,fenoterol, bitolterol. To LABAs belong compounds such as salmeterol,formoterol, indacaterol.

Whilst it is also known that β₂ adrenoceptor agonists providesymptomatic relief of patients suffering from asthma or COPD, anothercomponent of these airway disorders, i.e. inflammation often requiresseparate treatment. Typically this may involve treatment with aglucocorticoid, a LTRA or a PDE4 inhibitor, respectively. Currentlyavailable glucocorticoids include beclomethasone, triamcinolone,budesonide, fluticasone, mometasone, ciclesonide, loteprednol,etiprednol, flunisolide. LTRAs include pranlukast, montelukast,zafirlukast. PDE4 inhibitors are now under clinical development. Some ofthem are shortly before approval, e.g. roflumilast, AWD-12 281.

Bronchoconstriction and inflammation are also associated with increasedbronchial mucus production and possible bronchial plugging, which can betreated with anticholinergics, such as ipratropium, oxitropium,tiotropium, glycopyrrolate and in particular the RR enantiomer ofglycopyrrolate.

Bronchodilators (β₂-adrenoceptor agonists and anticholinergics) are nowthe mainstay of symptomatic therapy. Short- and long-actingβ₂-adrenoceptor agonists such as salbutamol, fenoterol, salmeterol,formoterol are established therapeutics in the symptomatic COPDmanagement. Of the short-acting antimuscarinic drugs, ipratropium iswidely used although antimuscarinic drugs are rather less efficacious inthe relief of an asthma attack than the β₂-adrenoceptor agonists(Rodrigo and Rodrigo, Chest 2003; 123:1908-15). Recently, tiotropium, along-acting anticholinergic with a certain preference to M₃-muscarinicreceptors has now been introduced world-wide (Hansel and Barnes, DrugsToday (Barc) 2002; 38:585-600, Koumis and Samuel, Clin Ther 2005;27:377-92). Relatively sufficient anti-inflammatory therapy withglucocorticoids are available for asthma. However, they are lesseffective in COPD. Additionally, tiotropium reduces COPD exacerbationsand related hospitalisations compared to placebo and ipratropium (Barret al. Cochrane Database Syst Rev 2005; (2):CD002876). Furtherlong-acting anticholinergics are now under development. They are moreconvenient to use for patients with advanced disease who requiremaintenance therapy with bronchodilators, and have been shown to providesuperior efficacy compared with short-acting antimuscarinic agents.

The basic tenet of asthma therapy has centred on optimal management ofthe underlying inflammatory process. The most recent expertrecommendations strongly support the use of inhaled glucocorticoids.Bronchodilators are used for transient relief of symptoms in thesepatients and for additional symptomatic control in patients who havepersistent symptoms despite inhaled glucocorticoid therapy(http://www.ginasthma.com/). In well-defined COPD, therapeuticalternatives vary. Because there is no currently accepted pharmacologictherapy that alters the natural history of the disease, therapeuticemphasis is placed on symptom relief. Unlike in asthma therapy,bronchodilators play a prominent role in the initial therapy forsymptomatic COPD patients. In this regard, long-acting β₂-adrenoceptoragonists and the new long-acting anticholinergic agent (tiotropium)offer distinct advantages. In contrast, inhaled glucocorticoids appearto have a more limited role in COPD. On the other hand, inhaledglucocorticoids have been shown to have beneficial effects on healthstatus and to decrease exacerbation rates in COPD patients.

Autoimmune diseases (e.g. rheumatoid arthritis [RA], systemic lupuserythematosus [SLE], multiple sclerosis [MS], glomerulonephritis,inflammatory bowel diseases [Morbus Cohn, colitis ulcerosa], psoriasis)belong to the major health problems worldwide. Autoimmune diseases areamong the ten leading causes of death. Most of autoimmune diseasesrequire lifelong treatment (Jacobson D L, Gange S J, Rose N R, Graham NM. Clin Immunol Immunopathol 1997; 84:223-43).

Autoimmune diseases are disorders in which the immune system producesantibodies against the body's own cells, destroying healthy tissue.These misdirected immune responses are referred to as autoimmunity,which can be demonstrated by the presence of autoantibodies or Tlymphocytes reactive with host antigens. Human autoimmune diseases canbe divided into two categories: organ-specific and systemic. Inorgan-specific autoimmune disease (e.g. Graves' disease, Hashimoto'sthyroiditis, type 1 diabetes mellitus), autoreactivity is directed toantigens unique to a single organ. In systemic autoimmune disease (e.g.RA, MS, SLE), autoreactivity is largely directed toward a broad range ofantigens and involves a number of tissues.

Systemic lupus erythematosus (SLE) is a chronic, usually life-long,potentially fatal autoimmune disease. SLE is notable for unpredictableexacerbations and remissions and a predilection for clinical involvementof the joints, skin, kidney, brain, lung, heart, and gastrointestinaltract.

Rheumatoid arthritis (RA), juvenile idiopathic arthritis, theseronegative spondylo-arthropathies including psoriatic arthritis, andsystemic lupus erythematosus are all examples of rheumatic diseases inwhich inflammation is associated with skeletal pathology. RA is the mostcommon inflammatory joint disease and a major cause of functionaldisability, morbidity, and premature mortality. Inflammation of, thesynovial membrane surrounding a joint leads to swollen, tender, andstiff joints. This may be accompanied by fatigue, weight loss, anxiety,and depression.

Rheumatoid arthritis (RA) usually requires lifelong treatment.Nonsteroidal anti-inflammatory drugs (NSAIDs) have been the cornerstoneof therapy for RA. NSAIDs reduce pain and inflammation and allow forimprovements in mobility and function. Aspirin and other NSAIDs, such asibuprofen, diclofenac, fenoprofen, indomethacin, naproxen are stronglyanti-flammatoric and analgetic. Their strong gastrointestinal sideeffects often limit their use. Cyclooxygenase-2 (COX-2) inhibitors wereinitially believed to cause fewer stomach problems than classicalNSAIDs. However, the use of COX-2 inhibitors is considerably limited duetheir cardiovascular undesired effects. Corticosteroids, used alone orin conjunction with other medications, may reduce the symptomatologyassociated with RA. In fact, corticosteroids reduce inflammation in RA.However, because of potential long-term side effects (e.g.gastrointestinal ulceration, osteoporosis, cataract development,glaucoma, etc.), corticosteroid use is usually limited to short coursesand low doses where possible. Drugs like anticholinergics are frequentlyused in combination with GCs to reduce ulcer development induced by GCs.

The current standard of care is aggressive therapy withanti-inflammatory drugs and disease-modifying anti-rheumatic drugs(DMARDs).

DMARDs most frequently are used in various combination therapy regimens.

Tumor necrosis factor (TNF) inhibitors are a relatively new class ofmedications used to treat autoimmune disease. They include etanercept(soluble TNF receptor), infliximab (monoclonal antibody against TNF),adalimumab (human TNF antiobody) and anakinra (recombinant humaninterleukin-1 receptor antagonist).

The term inflammatory bowel disease (IBD) describes a group of chronicinflammatory disorders of unknown causes involving the gastrointestinaltract (GIT). Patients with IBD can be divided into two major groups,those with ulcerative colitis (UC) and those with Crohn's disease (CD).In patients with UC, there is an inflammatory reaction primarilyinvolving the colonic mucosa. The inflammation is typically uniform andcontinuous with no intervening areas of normal mucosa. CD differs fromUC in that the inflammation extends through all layers of the intestinalwall and involves mesentery as well as lymph nodes. CD can occuranywhere along the intestinal tract.

Current therapies comprise aminosalicylates, drugs that contain5-aminosalicyclic acid (5-ASA), which help to control inflammation.Sulfasalazine is mainly useful in colonic disease because the activecompound, 5-aminosalicylic acid (5-ASA), is released in the large bowelby bacterial degradation of the parent compound. Products such asmesalamine that release 5-ASA in the distal small bowel secondary to pHchanges are more useful in patients with small intestinal Crohn disease.Other 5-ASA agents, such as olsalazine, mesalamine, and balsalazide,have a different carrier, fewer side effects, and may be used by peoplewho cannot take sulfasalazine. Immunomodulators such as azathioprine and6-mercapto-purine reduce inflammation by affecting the immune system.

Since TNF is a key inflammatory cytokine and mediator of intestinalinflammation, compounds directed against this cytokine such asinfliximab are promising in IBD. In certain cases, immunosuppressiveagents: cyclosporine A, tacrolimus, mycophenolate mofetil may beeffective in treating IBD.

GCs such as prednisone, methylprednisone, and hydrocortisone also reduceinflammation. They can be given orally, intravenously, through an enema,or in a suppository, depending on the location of the inflammation.These drugs can cause side effects such as gastrointestinal ulceration,weight gain, acne, facial hair, hypertension, diabetes, mood swings,bone mass loss, and an increased risk of infection. For this reason,they are not recommended for long-term use, although they are consideredvery affective when prescribed for short-term use.

In patients who relapse after withdrawal of steroids, other treatmentoptions are required. Steroids are not indicated for maintenance therapybecause of serious complications such as aseptic necrosis of the hip,osteoporosis, cataract, diabetes, and hypertension.

Multiple sclerosis (MS) is a chronic, potentially debilitating diseasethat affects the central nervous system characterized by the destructionof the myelin sheath surrounding neurons, resulting in the formation of“plaques”. MS is a progressive and usually fluctuating disease withexacerbations and remissions over many decades. MS affects more than 1million people around the world.

Current therapies target the immune dysfunction in MS and resultantneural tissue damage with the goal of preventing or at least reducingthe long-term risk of clinically significant disability. These therapiesare currently available: interferon(IFN)-β and glatiramer acetate(Copaxone®) (Linker R A, Stadelmann C, Diem R, Bahr M, Bruck W, Gold R.Fortschr Neurol Psychiatr 2005; 73:715-27; Strueby L, Nair B, Kirk A,Taylor-Gjevre R M. J Rheumatol 2005; 34:485-8). All of these medicationsappear to modulate the immune response in MS, although glatirameracetate and IFN medications probably work through different mechanisms.Each of these treatments (interferons and glatiramer acetate) offerdifferent advantages and disadvantages. Great disadvantages are route ofadministration by injection and the high costs.

The key to successful treatment of MS is to slow the inflammatoryprocess early in the disease. In order to prevent progression ofdisability, mitoxantrone, cyclophosphamide, methotrexate, azathioprineand cyclosporine are applied. GCs (e.g. methylprednisolone) are alsofrequently used. There is evidence that GCs slow down the brain atrophyprogression in MS patients (Zivadinov R. J Neurol Sci 2005; 233:73-81.

Psoriasis is a chronic, recurrent autoimmune disease of variableseverity that is considered to be a genetically programmed disease ofdysregulated inflammation, which is driven and maintained by multiplecomponents of the immune system.

Although topical drug preparations are usually sufficient to controlpsoriasis symptoms in patients with relatively mild disease, patientswith moderate to severe disease usually require phototherapy or systemicdrugs. The treatment of psoriasis includes ultraviolet,photochemotherapy, CsA, methotrexate and oral retinoid therapy (Naldi L,Griffiths C E. Br J Dermatol 2005; 152:597-615). There is alsoconsiderable experience supporting the efficacy of systemic therapy forpsoriasis (Bissonnette R. Skin. Therapy Lett 2006; 11:1-4). Manydifferent treatment options are available to control the symptoms ofpsoriasis. Mild to moderate cases are often treated first with topicaltreatments, such as coal tar, calcipotriol, salicylic acid, GCs.However, as the disease becomes more severe, treatment options such asphototherapy, systemic medication, and new biologics are employed. Somecommon systemic medications include: methotrexate, cyclosporine A,retinoids, vitamin A derivatives. Biologic medications, such as T-cellmodulators and TNF inhibitors (e.g. infliximab, alefacept, efalizumabetanercept), offer an alternative to other advanced therapies likesystemic medications and phototherapy (Menter A, Cather J C, Baker D,Farber H F, Lebwohl M, Darif M. J Am Acad Dermatol 2006; 54:61-3; Papp KA, Miller B, Gordon K B, Caro I, Kwon P, Compton P G, Leonardi C L; J AmAcad Dermatol 2006; 54(4 Suppl 1):S164-70).

Besides the mentioned drug classes, there are several novel drugs withinteresting modes of actions are now in clinical development for thetreatment of autoimmune diseases.

Current therapies for autoimmune diseases are not cures, but are aimedat reducing symptoms to provide the patient with an acceptable qualityof life. In organ-specific autoimmune disorders, symptoms can becorrected by metabolic control with biologically active compounds. Forexample, hypothroidism can be controlled by the administration ofthryroxine or diabetes mellitus can be treated with injections ofinsulin. Drugs used in most cases of autoimmune disease, especiallysystemic autoimmune disease, provide general nonspecific suppression ofthe immune system. For the most part these drugs do not distinguishbetween the pathological immune response and the protective immuneresponse. Immunosuppressive drugs (e.g., GCs, azathioprine,cyclophoshamide, CsA) are often given to suppress the proliferation ofautoreactive lymphocytes. Anti-inflammatory drugs also are prescribed topatients with RA. Unfortunately these drugs, besides not working in manypatients, have very serious side-effects. The general suppression of theimmune response puts the patient at greater risk to infection andcancer. Additionally, NSAIDs are effective in the pain management, theiruse may be associated with serious systemic adverse effects,particularly gastrointestinal disorders. Therefore, current treatmentsfor autoimmune diseases are not satisfactory. Given the high prevalenceof these diseases, improved, more effective and more convenienttherapeutic interventions are highly desirable. Clearly there is asignificant need for agents capable of treating autoimmune diseases. Itwould be desirable if such therapy could be administered in acost-effective and timely fashion, with a minimum of adverse sideeffects.

In general, treatment modalities in the therapy of autoimmune diseasesinclude symptomatic treatments using anti-inflammatory drugs,immunosuppressant medications, immunomodulating therapy and others. Nosingle regimen is appropriate. Therefore, combinations of differentmedications are preferred.

Various approaches have been taken to suppress this inappropriateautoimmune inflammatory response. The efficacy of different classes ofdrugs, e.g. GCs, PDE4 inhibitors and others have shown only limitedefficacy, perhaps because blocking only one of many pathways does notprovide a sufficiently large decrease in overall inflammatory processes.The other approach is the use of a combination of drugs which targetseveral pathogenetical processes.

Glycopyrrolate belongs to the so-called anticholinergic drugs andantagonizes the neurotransmitter acetylcholine at its receptor site.This effect leads to a considerable bronchodilatation and a reducedmucus secretion. Long-acting β₂-adrenoceptor agonists (LABA) (e.g.salmeterol, formoterol, indacaterol) are the basis therapy of asthma andfrequently in COPD. They stimulate the adenylate cyclase and increasethe intracellular cAMP content resulting in bronchodilation. Antagonistsof BLT- and CysLT-receptors reduce the inflammatory processes in theairways. Thus, these effects of LT-receptor-antagonists result in animproved mucosal and bronchial function in patients suffering frombronchial asthma or COPD. To date, one of the most effective medicationsfor airway inflammation is inhaled glucocorticoids (GCs). Thesecompounds practically inhibit all important inflammatory processesinvolved in asthma and partly in COPD, at least, to a considerableextent. Inhibitors of the isoenzyme PDE4 reduce the inflammatoryprocesses both in asthma and COPD.

A cornerstone in the management of asthma and COPD is the control ofairway inflammation. All drug classes mentioned above are able to reduceairway inflammation to various extents. Therefore, our intention was tocombine these drug classes with the aim to improve the bronchodilatoryeffect and to enhance the anti-inflammatory activity.

The pharmacodynamic properties of all these drug classes,anticholinergics (especially R,R-glycopyrrolate), β₂-adrenoceptoragonists, antagonists of BLT- and CysLT-receptors, PDE4 inhibitors,and/or inhaled GCs complement one another and result in more efficacioustreatment of the mentioned diseases, which is surprisingly over-additiveeven compared to the dual combinations. Furthermore, since the doses ofmonocompounds are in the combination lower, the incidence of adverseeffects can be reduced by using the combination. Additionally, thepatients' compliance is also increased.

Both short- and long-acting β₂-adrenoceptor agonists play a pivotal rolein the acute and chronic management of asthma. They β₂-adrenoceptoragonists are an integral part of the treatment in COPD and asthma, andcombination of two or more bronchodilator agents generally is needed tocontrol symptoms. Patients with either condition can benefit frombronchodilators. Their major action on the airways is the relaxation ofsmooth muscle cells. In addition to their bronchodilator properties,β₂-adrenoceptor agonists may have other effects through their activationof β₂-adrenoceptors expressed on resident airway cells such asepithelial cells and mast cells and circulating inflammatory cells suchas eosinophils and neutrophils. These non-bronchodilator activities ofβ₂-adrenoceptor agonists may enhance their efficacy in the management ofasthma. In pre-clinical studies, the anti-inflammatory effects ofβ₂-adrenoceptor agonists are demonstrated through their stabilizingeffect on mast cells and their inhibition of mediator release fromeosinophils, macrophages T-lymphocytes, and neutrophils. In addition,β₂-adrenoceptor agonists may inhibit plasma exudation in the airway, andmediator release from epithelial cells.

The regular use of short-acting β₂-adrenoceptor agonists alone has beenshown to have deleterious effects on asthma control. Therefore,short-acting agents should only be used when needed for rescue of acutesymptoms. Monotherapy with long-acting β₂-adrenoceptor agonists (LABAs)has also been associated with poor asthma control. However, when givenconcomitantly with inhaled glucocorticoids, β₂-adrenoceptor agonists maypotentiate the anti-inflammatory effect of glucocorticoid, improveasthma control and prevent exacerbations (Hanania and Moore, Curr DrugTargets Inflamm Allergy 2004; 3:271-7). Basically, the combination ofLABAs and inhaled GCs is useful in both conditions.

Antimuscarinic treatment of asthma and COPD has a relatively longhistory leading to its present day use as an effective bronchodilatingdrug for obstructive pulmonary diseases. Anticholinergic agents areexemplified by the belladonna alkaloids atropine and scopolamine, whichinhibit the muscarinic action of acetylcholine on structure innervatedby postganglionic cholinergic nerves. These agents typically inhibitbronchoconstriction by relaxing of smooth muscles and cause considerablebronchodilation. Anticholinergic agents also are known to exert centraleffects which include pupil dilatation and stimulation and/or depressionof the central nervous system. Novel anticholinergic pharmaceuticalshave been developed which have a limited capacity to pass across theblood-brain barrier, and therefore have a limited capacity to producecentral effects. Examples of these agents are the quaternary ammoniumcompounds methscopolamine, ipratropium, oxitropium, tiotropium and theenantiomers of glycopyrrolate. Some experts recommend ipratropium as thefirst choice. It has a very slow onset and can be used as maintenancetherapy for people with emphysema and chronic bronchitis with few severeside effects. However, a patient should not take more than 12inhalations per day. The recently introduced tiotropium has a longduration of action and superior to ipratropium.

Anticholinergic medications have been accepted as an important treatmentmodality in COPD and chronic asthma. The anticholinergic bronchodilator,the muscarinic receptor antagonist, used in this invention will be along-acting compound. Any compound of this type can be used in thiscombination therapy approach. By long-lasting it is meant that the drugwill have an effect on the bronchi that lasts around 12 hours or more,up to 24 hours. The recently approved long acting inhaledanticholinergic drug, tiotropium, produces sustained bronchodilationthroughout the 24 hour day (Calverley et al. Thorax 2003a; 58:855-60).In fact, bronchodilators improve symptoms and quality of life, in COPDpatients, but, with the exception of tiotropium, they do notsignificantly influence the natural course of the disease (Caramori andAdcock, Pulm Pharmacol Ther 2003; 16:247-77).

Glycopyrrolate, a quaternary ammonium anticholinergic compound, consistsof four stereoisomers. It is poorly absorbed from mucus membranes, thusreducing anticholinergic side effects (Ali-Melkkila et al. ActaAnaesthesiol Scand 1993; 37:633-42). Glycopyrrolate possesses noselectivity in its binding to the M₁-M₃ receptors. Kinetics studies,however, showed that glycopyrrolate dissociates slowly from M₃muscarinic receptors (Haddad et al. Br J Pharmacol 1999; 127:413-20).Similarly to tiotropium, this behavior explains glycopyrrolate'srelative receptor selectivity and its long duration of action. Indeed,there is evidence that racemic glycopyrrolate produces considerable andlong-lasting bronchodilatory effects both in asthmatic and in COPDpatients (Walker et al. Chest 1987; 91:49-51, Schroeckenstein et al. JAllergy Clin Immunol 1988; 82:115-9, Gilman et al. Chest 1990;98:1095-8, Cydulka and Emerman, Ann Emerg Med 1995; 25:470-3, Hansel etal. Chest 2005; 128:1974-9). As asthma and COPD are characterized byincreased mucus secretions, the antisecretory effect of anticholinergicssuch glycopyrrolate is an additional advantage for their use in thetherapy of these diseases.

A cornerstone in the management of asthma and COPD is the control ofairway inflammation.

Inhaled glucocorticoids (GCs) are the most effective long-term therapyin controlling chronic asthma symptoms (Barnes Ernst Schering Res FoundWorkshop. 2002; 40:1-23). Randomized, controlled clinical studiesconfirm the efficacy of early intervention with inhaled glucocorticoidsin patients with mild persistent asthma (Sheffer et al. Ann AllergyAsthma Immunol 2005; 94:48-54). Inhaled glucocorticoids suppresseosinophilic inflammation in the airways and are a corner-stone inasthma treatment (van Rensen et al. Thorax 1999; 54:403-8, Barnes ErnstSchering Res Found Workshop. 2002; 40:1-23). However, patients show avariable response to inhaled glucocorticoids and some exhibitglucocorticoid resistance.

In COPD the airway inflammation is quite different from asthma, soinflammation and anti-inflammatory treatment in COPD is relatively a newfocus of interest. In contrast to asthma, the practice of using inhaledglucocorticoids in COPD is common but controversial (Crapo et al. EurRespir J Suppl 2003; 41:19s-28s, O'Riordan, J Aerosol Med 2003; 16:1-8).It is likely that inhaled GCs have little effect on the progression ofCOPD (Vonk et al. Thorax 2003; 58, 322-327). glucocorticoids areprobably scarcely effective in COPD patients without overlappingconcomitant asthma. The use of inhaled glucocorticoids in patients withmoderately severe disease (FEV₁<50% predicted) may produce clinicalbenefit as measured by an increase in FEV₁, reduced symptoms and fewerexacerbations (O'Riordan, J Aerosol Med 2003; 16:1-8). However, there isalso clinical evidence that the neutrophil inflammation may be decreasedby inhaled glucocorticoids in clinically stable COPD patients (Yildiz etal. Respiration 2000; 67:71-6). Consequently, in a recently publishedguideline on COPD treatment, adding on inhaled glucocorticoid isrecommended in moderate to severe disease (Pauwels et al. Am J RespirCrit Care Med 2001; 163:1256-76). glucocorticoids should mainly be usedto reduce exacerbations and improve the health status of these patients(Nishimura et al. Chest 1999; 115:31-7, Selroos, Curr Med Res Opin 2004;20:1579-93). But it has to be admitted that current pharmacologicaltreatment of COPD is unsatisfactory, as it does not significantlyinfluence the severity of the disease or its natural course. In general,inhaled glucocorticoids have relatively little impact on theinflammatory processes that characterize COPD (Adcock and Chung, CurrOpin Investig Drugs 2002; 3:58-60) and are not suitable for monotherapyin COPD but can be helpfully combined with an inhaled bronchodilator(Calverley et al. Eur Respir J 2003b; 22:912-9, Calverley Semin RespirCrit Care Med 2005; 26:235-45). They have, however, been shown todecrease the frequency of exacerbations and improve quality of life inpatients with COPD (Calverley Semin Respir Crit Care Med 2005;26:235-45).

The administration of inhaled glucocorticoids or anticholinergics (e.g.ipratropium) reduced the risk of hospitalization of COPD patients(Rascati et al. Clin Ther 2005; 27:346-54). The combination oflong-acting muscarinic antagonists and inhaled glucocorticoids is moreefficacious in asthma and COPD than either alone. Indeed, there isevidence that corticosteroid/muscarinic antagonist combination therapyhas complementary, additive, or synergistic inhibitory effects onproinflammatory signalling pathways, inflammatory mediator release, andrecruitment and survival of inflammatory cells. In the patient with anairway inflammatory disease such as asthma or COPD, this is reflected inenhanced anti-inflammatory activity with combination therapy beyond thatwhich can be achieved by either drug alone, or the potential forantimuscarinic drugs to provide a steroid-sparing effect.

Leukotriene receptor antagonists (LTRA) are a relatively newanti-inflammatory class of anti-asthma drugs. Leukotrienes (LTs) andtheir receptors play an important role in the pathogenesis of asthma,and they are also involved in COPD. More recently, the potentialinvolvement of the monocyte-macrophage lineage in the etiology of COPDhas received growing attention as a target for leukotriene inhibition(Kilfeather Chest 2002; 121(5 Suppl):197S-200S). The main effectsmediated via LTs are bronchoconstriction, airway inflammation, edema andmucus hypersecretion. Arachidonic acid metabolism via 5-lipoxygenase(5-LOX) results in a group of biologically active lipids known as LTs.LTB₄ is a potent activator of leukocyte chemotaxis. Cysteinyl-LTs (LTC₄,LTD₄, LTE₄) account for the spasmogenic activity previously described asslow-reacting substance of anaphylaxis (SRS-A). These inflammatorymediators are produced by a number of cell types including mast cells,neutrophils, eosinophils, basophils, macrophages and monocytes. Theyexert their biological effects by binding and activating specificreceptors (LTB₄ at the BLT receptor, cysteinyl-LTs at thecysLT₁-receptor). This occurs in a series of events that lead tocontraction of the human airway smooth muscle, chemotaxis and increasedvascular permeability, mucus hypersecretion, decrease of ciliarymotility. These effects have led to their important role in the diseasesof asthma, allergic rhinitis and COPD. CysLT-receptor antagonists(zafirlukast, montelukast and pranlukast) represent an effective andwell-tolerated treatment for asthma in adults and children, particularlyfor exercise- and aspirin-induced asthma. According to currentguidelines for asthma management, anti-inflammatory therapy with inhaledglucocorticoids is the cornerstone in the treatment of persistentasthma. To further optimize asthma control, add-on therapy with LABA orLTRA should be combined with low to high doses of inhaledglucocorticoids. While the first combination focuses on optimal controlof symptoms and lung function, the second provides a more completesuppression of the airway inflammation. They can also have clinicalapplications in the COPD. Recently, it has been suggested thatzafirlukast, a cysLT-receptor antagonist may increase the tidal volumeand alveolar ventilation in patients suffering from COPD (Bu et al. ChinMed J 2003; 116:459-461).

Even if there are no compelling clinical data for an additionalcontribution by LTB₄ in human asthma, in other respiratory conditionssuch as COPD, which are characterised by pronounced neutrophilinfiltration, it may be that the chemotactic properties of LTB₄ are moreimportant (Daniel and O'Byrne, Am Rev Respir Dis 1991; 143:S3-5). Inpatients suffering from COPD, the enhanced oxidative stress isparalleled by the increased ability of neutrophil to synthesize thechemotactic factor LTB₄, and may ultimately contribute to theinfiltration/activation of neutrophils into the airways of COPD patients(Santus et al. Am J Respir Crit Care Med 2005; 171:838-43).Additionally, there is a selective increase in exhaled LTB₄ in patientswith COPD (Montuschi et al. Thorax 2003; 58:585-8).

Cyclic adenosine monophosphate (adenosine 3′,5′-cyclic monophosphate,[cAMP]) is known as a second messenger that mediates cellular responsesto several compounds e.g. hormones, mediators, etc. Phosphodiesterases(PDEs) are a family of enzymes that metabolize 3′,5′ cyclic nucleotidesto 5′ nucleoside monophosphates, thereby terminating cAMP and cGMPsecond messenger activity. A particular PDE, PDE4, which is a highaffinity, cAMP specific, type 4 PDE, has generated interest as potentialtargets for the development of novel anti-inflammatory compounds. Infact, PDE4 regulate intracellular levels of cAMP and are the predominantPDE expressed in inflammatory cells. Inhibitors of PDE4 act byincreasing intracellular concentrations of cyclic AMP, which has a broadrange of anti-inflammatory effects on various key effector cellsinvolved in asthma and COPD (Barnette et al. J Pharmacol Exp Ther 1998;284:420-6, Hatzelmann and Schudt, J Pharmacol Exp Ther 2001; 297:267-79,Marx et al. Pulm Pharmacol Ther 2002; 15:7-15, Kuss et al. J PharmacolExp Ther 2003; 307:373-85). They show a broad spectrum of activity inanimal models of asthma COPD (Howell et al. 1995, Bundschuh et al. 2002,Billah et al. 2002, Kuss et. al. J Pharmacol Exp Ther 2003; 307:373-85).In addition, activation of the cAMP signalling pathway in airway smoothmuscle cells promotes relaxation and blocks smooth muscle cellreplication (Tomlinson et al. Biochem Pharmacol 1995; 49: 1809-19), thuspreventing the airway remodelling observed in the chronic stage of thediseases. Studies on cilomilast, roflumilast and other PDE4 inhibitorsin asthma and COPD have shown a broad range of anti-inflammatoryactivity, and the available evidence on clinical outcomes (Compton etal. Lancet 2001; 358:265-7, Dyke and Montana, Expert Opin Investig Drugs2002; 11:1-13, Grootendorst et al. Pulm Pharmacol Ther 2003; 16:341-7,Spina Curr Drug Targets Inflamm Allergy 2004; 3:231-6, Lipworth Lancet2005; 365:167-75, Bäumer et al. Exp Rev 2005; 1:134-45, Rabe et al.Lancet 2005; 366:563-71). Efforts to minimize or eliminate theabove-mentioned adverse events sometimes associated with PDE4 inhibitorshave included creating inhibitors which do not penetrate the centralnervous system, and administering PDE4 inhibitors by inhalation ratherthan orally. It is likely that these class-associated side effects,mainly nausea and emesis, could be at least partially overcome by theso-called “second-generation” PDE4 inhibitors that can be applied byinhalation.

The PDE4 inhibitor useful in this invention may be any compound that isknown to inhibit the PDE4 enzyme and which is discovered to act ashighly specific PDE4 inhibitors and which is preferably used perinhalationem. For example, preclinical and clinical studies with thehighly potent and selective PDE4 inhibitor AWD 12-281 showed that thiscompound has a good preclinical and clinical efficacy. In Brown Norwayrats, AWD 12-281 suppressed allergen-induced airway eosinophilia with anID₅₀ of 7 μg/kg when administered directly into the lungs. TheID₅₀-value of the known glucocorticoid beclomethasone was comparable(0.1 μg/kg). Due to its unique metabolic profile, the compound has asuitable safety profile after topical (nasal or inhaled) administration.When AWD 12-281 given to dogs by inhalation, no emesis could be inducedup to the highest feasible dose (15 mg/kg) indicating that AWD 12-281 isuseful for the inhaled treatment of asthma and COPD (Kuss et al. JPharmacol Exp Ther 2003; 307:373-85).

Inhibition of PDE4 results in an elevation of cAMP in the inflammatorycells, which in turn downregulates the inflammatory response. Forexample, rolipram, a PDE4 inhibitor, reduced the clinical andhistological severity of collagen-induced arthritis in rats (Nyman U,Mussener A, Larsson E, Lorentzen J, Klareskog L: Amelioration ofcollagen II-induced arthritis in rats by the type IV phosphodiesteraseinhibitor rolipram.). It has also been demonstrated that selective PDE4inhibition suppresses the clinical manifestations of EAE (Sommer N,Martin R, McFarland H F, Quigley L, Cannella B, Raine C S, Scott D E,Loschmann P A, Racke M K. Therapeutic potential of phosphodiesterasetype 4 inhibition in chronic autoimmune demyelinating disease. JNeuroimmunol 1997; 79:54-61). PDE4 inhibitors might also havetherapeutic benefit both in IBD (Banner K H, Trevethick M A. PDE4inhibition: a novel approach for the treatment of inflammatory boweldisease. Trends Pharmacol Sci 2004; 25:430-6), and in psoriasis (HouslayM D, Schafer P, Zhang K Y. Keynote review: phosphodiesterase-4 as atherapeutic target. Drug Discov Today 2005; 10:1503-19). One of thepossible side effects induced by PDE4 inhibitors may be a certainsedation, decrease in locomotor activity. In animal experiments, it hasconvincingly been demonstrated that rolipram, a specific PDE4 inhibitorinduces sedative effects such as hypoactivity, decreased locomotion thatwere completely reversed by adding scopolamine, an anticholinergiccompound (Silvestre et al. Pharmacol Biochem Behav 1999; 64:1-5).Consequently, an antimuscarinic agents may compensate the possiblesedation induced by a PDE4 inhibitor and thus improve the therapeuticvalue of the combination.

It is well known that inhaled glucocorticoids represent a first choicepharmacological intervention in the therapy of inflammatory respiratorydiseases. This class of drugs, among which can be mentioned for exampletriamcinolone, beclomethasone, mometasone, fluticasone, budesonide,etc., elicits remarkable pharmacodynamics effects on the respiratorytract. Additionally, they also elicit undesired effects on differentorgans, and for this reason both their clinical use and its interruptioncause a series of side effects, some of which very serious.

Among said toxic effects can be mentioned those affecting the bonetissue leading to an altered cellular metabolism and a high osteoporosisincidence. Several studies showed that inhaled glucocorticoids cause areduction in bone mineral density leading to an increase in fractures inpeople taking an inhaled glucocorticoid compared with controls (Ip etal. Chest 1994; 1051722-7, Mortimer et al. Ann Allergy Asthma Immunol2005; 94:15-21). Strategies are needed to reduce the systemic effects ofinhaled glucocorticoids. Inhibition of PDE4 activity is believedeffective for the treatment of osteoporosis by reducing bone loss. Thereis evidence that the PDE4 isoenzyme may play an important role in boneturnover through cAMP and that its inhibitors are candidates fortherapeutic drugs for the bone loss diseases (Miyamoto et al. BiochemPharmacol 1997; 54:613-7). Indeed, rolipram can enhance physiologicalbone formation and thereby increase bone mass in mice (Kinoshita et al.Bone 2000; 27:811-7). PDE4 inhibitors are apparently able to counteractthe bone-demineralising effect of glucocorticoids. Therefore, it wouldbe desirable to provide novel compositions that contain besidesglucocorticoids PDE4 inhibitors as well.

There is evidence that inhaled glucocorticoids are especiallyadvantageous in combination with bronchodilators (Donohue et al. TreatRespir Med 2004; 3:173-81). Furthermore, it has also been demonstratedthat β₂-adrenoceptor agonists added to the existing therapy regimenconsisting of an anticholinergic (ipratropium) and an inhaledglucocorticoid (beclomethasone) provides greater symptomatic relief andimprovement in lung function than placebo (Gupta and Chhabra, Indian JChest Dis Allied Sci 2002; 44:165-72).

Current treatments for asthma and COPD are not satisfactory. Given thehigh prevalence of these diseases, improved, more effective and moreconvenient therapeutic interventions are highly desirable.

Therefore the problem underlying the invention was to present apharmaceutical, which is improved for the treatment of inflammatorydiseases especially respiratory diseases as asthma or COPD, but also forrheumatism or auto-immune diseases.

This problem is solved by a combination of at least three differentpharmaceutically active substances or their physiologically acceptablesalts selected from the group of anticholinergics, PDE4 inhibitors,glucocorticoids, β₂-adrenoceptor agonists and Leukotriene-receptorantagonists, which show higher efficiency and reduced side effectscompared to the single substances or double combinations which could notbe expected from prior art.

The combinations comprise at least three different pharmaceuticallyactive substances or their pharmaceutically acceptable salts selectedfrom of the following groups:

A: anticholinergics: 1: methscopolamine, 2: ipratropium, 3: oxitropium,4: tiotropium 5: racemic glycopyrrolate, 6: R,R-glycopyrrolateB: PDE 4 Inhibitors 1: rolipram, 2: roflumilast, 3: cilomilast, 4:AWD-12-281C: Glucocorticoids: 1: budesonide, 2: fluticasone, 3: mometasone, 4:beclomethasone, 5: ciclesonide, 6: triamcinolone, 7: loteprednol, 8:etiprednol, 9: flunisolideD: β₂-adrenoceptor agonists 1: salbutamol, 2: terbutaline, 3:salmeterol, 4: formoterol, 5: indacaterol 6: fenoterol, 7: reproterol,8: pirbuterol, 9: bitolterolE: Leukotriene-receptor antagonists 1: pranlukast, 2: montelukast, 3:zafirlukast

Preferred combinations comprise three different pharmaceutically activesubstances or their pharmaceutically acceptable salts selected from thegroups of

Anticholinergics, PDE4 inhibitors and GlucocorticoidsAnticholinergics, PDE4 inhibitors and β₂-adrenoceptor agonistsAnticholinergics, PDE4 inhibitors and Leukotriene-receptor antagonistsAnticholinergics, Glucocorticoids, and β₂-adrenoceptor agonistsAnticholinergics, Glucocorticoids, and Leukotriene-receptor antagonistsPDE4 inhibitors, Glucocorticoids, and β₂-adrenoceptor agonistsPDE4 inhibitors, β₂-adrenoceptor agonists, and Leukotriene-receptorantagonistsGlucocorticoids, β₂-adrenoceptor agonists, and Leukotriene-receptorantagonists

Especially preferred combinations are comprising three differentpharmaceutically active substances or their pharmaceutically acceptablesalts selected from the groups of

R,R-glycopyrrolate, PDE4 inhibitors and GlucocorticoidsR,R-glycopyrrolate, PDE4 inhibitors and β₂-adrenoceptor agonistsR,R-glycopyrrolate, PDE4 inhibitors and Leukotriene-receptor antagonistsR,R-glycopyrrolate, Glucocorticoids, and β₂-adrenoceptor agonistsR,R-glycopyrrolate, Glucocorticoids, and Leukotriene-receptorantagonists

The following combinations show the effects according to the invention:

A1,B1, C1; A1,B1, C2; A1,B1, C3; A1,B1, C4; A1,B1, C5; A1,B1, C6; A1,B1,C7; A1,B1, C8; A1,B1, C9; A1,B2, C1; A1,B2, C2; A1,B2, C3; A1,B2, C4;A1,B2, C5; A1,B2, C6; A1,B2, C7; A1,B2, C8; A1,B2, C9;

A1,B3, C1; A1,B3, C2; A1,B3, C3; A1,B3, C4; A1,B3, C5; A1,B3, C6; A1,B3,C7; A1,B3, C8; A1,B3, C9;

A1,B4, C1; A1,B4, C2; A1,B4, C3; A1,B4, C4; A1,B4, C5; A1,B4, C6; A1,B4,C7; A1,B4, C8; A1,B4, C9; A2,B1, C1; A2,B1, C2; A2,B1, C3; A2,B1, C4;A2,B1, C5; A2,B1, C6; A2,B1, C7; A2,B1, C8; A2,B1, C9; A2,B2, C1; A2,B2,C2; A2,B2, C3; A2,B2, C4; A2,B2, C5; A2,B2, C6; A2,B2, C7; A2,B2, C8;A2,B2, C9; A2,B3, C1; A2,B3, C2; A2,B3, C3; A2,B3, C4; A2,B3, C5; A2,B3,C6; A2,B3, C7; A2,B3, C8; A2,B3, C9; A2,B4, C1; A2,B4, C2; A2,B4, C3;A2,B4, C4; A2,B4, C5; A2,B4, C6; A2,B4, C7; A2,B4, C8; A2,B4, C9; A3,B1,C1; A3,B1, C2; A3,B1, C3; A3,B1, C4; A3,B1, C5; A3,B1, C6; A3,B1, C7;A3,B1, C8; A3,B1, C9; A3,B2, C1; A3,B2, C2; A3,B2, C3; A3,B2, C4; A3,B2,C5; A3,B2, C6; A3,B2, C7; A3,B2, C8; A3,B2, C9; A3,B3, C1; A3,B3, C2;A3,B3, C3; A3,B3, C4; A3,B3, C5; A3,B3, C6; A3,B3, C7; A3,B3, C8; A3,B3,C9; A3,B4, C1; A3,B4, C2; A3,B4, C3; A3,B4, C4; A3,B4, C5; A3,B4, C6;A3,B4, C7; A3,B4, C8; A3,B4, C9; A4,B1, C1; A4,B1, C2; A4,B1, C4; A4,B1,C4; A4,B1, C4; A4,B1, C6; A4,B1, C7; A4,B1, C8; A4,B1, C9; A4,B2, C1;A4,B2, C2; A4,B2, C3; A4,B2, C4; A4,B2, C5; A4,B2, C6; A4,B2, C7; A4,B2,C8; A4,B2, C9; A4,B3, C1; A4,B3, C2; A4,B3, C3; A4,B3, C4; A4,B3, C5;A4,B3, C6; A4,B3, C7; A4,B3, C8; A4,B3, C9; A4,B4, C1; A4,B4, C2; A4,B4,C3; A4,B4, C4; A4,B4, C5; A4,B4, C6; A4,B4, C7; A4,B4, C8; A4,B4, C9;A5,B1, C1; A5,B1, C2; A5,B1, C3; A5,B1, C4; A5,B1, C5; A5,B1, C6; A5,B1,C7; A5,B1, C8; A5,B1, C9; A5,B2, C1; A5,B2, C2; A5,B2, C3; A5,B2, C4;A5,B2, C5; A5,B2, C6; A5,B2, C7; A5,B2, C8; A5,B2, C9; A5,B3, C1; A5,B3,C2; A5,B3, C3; A5,B3, C4; A5,B3, C5; A5,B3, C6; A5,B3, C7; A5,B3, C8;A5,B3, C9; A5,B4, C1; A5,B4, C2; A5,B4, C3; A5,B4, C4; A5,B4, C5; A5,B4,C6; A5,B4, C7; A5,B4, C8; A5,B4, C9; A6,B1, C1; A6,B1, C2; A6,B1, C3;A6,B1, C4; A6,B1, C5; A6,B1, C6; A6,B1, C7; A6,B1, C8; A6,B1, C9; A6,B2,C1; A6,B2, C2; A6,B2, C3; A6,B2, C4; A6,B2, C5; A6,B2, C6; A6,B2, C7;A6,B2, C8; A6,B2, C9; A6,B3, C1; A6,B3, C2; A6,B3, C3; A6,B3, C4; A6,B3,C5; A6,B3, C6; A6,B3, C7; A6,B3, C8; A6,B3, C9; A6,B4, C1; A6,B4, C2;A6,B4, C3; A6,B4, C4; A6,B4, C5; A6,B4, C6; A6,B4, C7; A6,B4, C8; A6,B4,C9; A1,B1, D1; A1,B1, D2; A1,B1, D3; A1,B1, D4; A1,B1, D5; A1,B1, D6;A1,B1, D7; A1,B1, D8; A1,B1, D9; A1,B2, D1; A1,B2, D2; A1,B2, D3; A1,B2,D4; A1,B2, D5; A1,B2, D6; A1,B2, D7; A1,B2, D8; A1,B2, D9; A1,B3, D1;A1,B3, D2; A1,B3, D3; A1,B3, D4; A1,B3, D5; A1,B3, D6; A1,B3, D7; A1,B3,D8; A1,B3, D9; A1,B4, D1; A1,B4, D2; A1,B4, D3; A1,B4, D4; A1,B4, D5;A1,B4, D6; A1,B4, D7; A1,B4, D8; A1,B4, D9; A3,B1, D1; A3,B1, D2; A3,B1,D3; A3,B1, D4; A3,B1, D5; A3,B1, D6; A3,B1, D7; A3,B1, D8; A3,B1, D9;

A3,B2, D1; A3,B2, D2; A3,B2, D3; A3,B2, D4; A3,B2, D5; A3,B2, D6; A3,B2,D7; A3,B2, D8; A3,B2, D9;

A3,B3, D1; A3,B3, D2; A3,B3, D3; A3,B3, D4; A3,B3, D5; A3,B3, D6; A3,B3,D7; A3,B3, D8; A3,B3, D9; A3,B4, D1; A3,B4, D2; A3,B4, D3; A3,B4, D4;A3,B4, D5; A3,B4, D6; A3,B4, D7; A3,B4, D8; A3,B4, D9; A4,B1, D1; A4,B1,D2; A4,B1, D3; A4,B1, D4; A4,B1, D5; A4,B1, D6; A4,B1, D7; A4,B1, D8;A4,B1, D9; A4,B2, D1; A4,B2, D2; A4,B2, D3; A4,B2, D4; A4,B2, D5; A4,B2,D6; A4,B2, D7; A4,B2, D8; A4,B2, D9; A4,B3, D1; A4,B3, D2; A4,B3, D3;A4,B3, D4; A4,B3, D5; A4,B3, D6; A4,B3, D7; A4,B3, D8; A4,B3, D9; A4,B4,D1; A4,B4, D2; A4,B4, D3; A4,B4, D4; A4,B4, D5; A4,B4, D6; A4,B4, D7;A4,B4, D8; A4,B4, D9; A5,B1, D1; A5,B1, D2; A5,B1, D3; A5,B1, D4; A5,B1,D5; A5,B1, D6; A5,B1, D7; A5,B1, D8; A5,B1, D9; A5,B2, D1; A5,B2, D2;A5,B2, D3; A5,B2, D4; A5,B2, D5; A5,B2, D6; A5,B2, D7; A5,B2, D8; A5,B2,D9; A5,B3, D1; A5,B3, D2; A5,B3, D3; A5,B3, D4; A5,B3, D5; A5,B3, D6;A5,B3, D7; A5,B3, D8; A5,B3, D9; A5,B4, D1; A5,B4, D2; A5,B4, D3; A5,B4,D4; A5,B4, D5; A5,B4, D6; A5,B4, D7; A5,B4, D8; A5,B4, D9; A6,B1, D1;A6,B1, D2; A6,B1, D3; A6,B1, D4; A6,B1, D5; A6,B1, D6; A6,B1, D7; A6,B1,D8; A6,B1, D9; A6,B2, D1; A6,B2, D2; A6,B2, D3; A6,B2, D4; A6,B2, D5;A6,B2, D6; A6,B2, D7; A6,B2, D8; A6,B2, D9; A6,B3, D1; A6,B3, D2; A6,B3,D3; A6,B3, D4; A6,B3, D5; A6,B3, D6; A6,B3, D7; A6,B3, D8; A6,B3, D9;A6,B4, D1; A6,B4, D2; A6,B4, D3; A6,B4, D4; A6,B4, D5; A6,B4, D6; A6,B4,D7; A6,B4, D8; A6,B4, D9; A1,B1, E1; A1,B1, E2; A1,B1, E3; A1,B1, E4;A1,B2, E1; A1,B2, E2; A1,B2, E3; A1,B2, E4; A1,B3, E1; A1,B3, E2; A1,B3,E3; A1,B3, E4; A1,B4, E1; A1,B4, E2; A1,B4, E3; A1,B4, E4; A2,B1, E1;A2,B1, E2; A2,B1, E3; A2,B1, E4; A2,B2, E1; A2,B2, E2; A2,B2, E3; A2,B2,E4; A2,B3, E1; A2,B3, E2; A2,B3, E3; A2,B3, E4; A2,B4, E1; A2,B4, E2;A2,B4, E3; A2,B4, E4; A3,B1, E1; A3,B1, E2; A3,B1, E3; A3,B1, E4; A3,B2,E1; A3,B2, E2; A3,B2, E3; A3,B2, E4; A3,B3, E1; A3,B3, E2; A3,B3, E3;A3,B3, E4; A3,B4, E1; A3,B4, E2; A3,B4, E3; A3,B4, E4; A4,B1, E1; A4,B1,E2; A4,B1, E3; A4,B1, E4; A4,B2, E1; A4,B2, E2; A4,B2, E3; A4,B2, E4;A4,B3, E1; A4,B3, E2; A4,B3, E3; A4,B3, E4; A4,B4, E1; A4,B4, E2; A4,B4,E3; A4,B4, E4; A5,B1, E1; A5,B1, E2; A5,B1, E3; A5,B1, E4; A5,B2, E1;A5,B2, E2; A5,B2, E3; A5,B2, E4; A5,B3, E1; A5,B3, E2; A5,B3, E3; A5,B3,E4; A5,B4, E1; A5,B4, E2; A5,B4, E3; A5,B4, E4; A6,B1, E1; A6,B1, E2;A6,B1, E3; A6,B1, E4; A6,B2, E1; A6,B2, E2; A6,B2, E3; A6,B2, E4; A6,B3,E1; A6,B3, E2; A6,B3, E3; A6,B3, E4; A6,B4, E1; A6,B4, E2; A6,B4, E3;A6,B4, E4; A1,C1,D1; A1,C1,D2; A1,C1,D3; A1,C1,D4; A1,C1,D5; A1,C1,D6;A1,C1,D7; A1,C1,D8; A1,C1, D9; A1,C2, D1; A1,C2,D2; A1,C2,D3; A1,C2,D4;A1,C2, D5; A1,C2, D6; A1,C2,D7; A1,C2,D8; A1,C2, D9; A1,C3, D1;A1,C3,D2; A1,C3,D3; A1,C3,D4; A1,C3, D5; A1,C3, D6; A1,C3,D7; A1,C3,D8;A1,C3, D9; A1,C4, D1; A1,C4,D2; A1,C4,D3; A1,C4,D4; A1,C4, D5; A1,C4,D6; A1,C4,D7; A1,C4,D8; A1,C4,D9; A1,C5, D1; A1,C5,D2; A1,C5,D3;A1,C5,D4; A1,C5, D5; A1,C5, D6; A1,C5,D7; A1,C5,D8; A1,C5, D9; A1,C6,D1; A1,C6,D2; A1,C6,D3; A1,C6,D4; A1,C6, D5; A1,C6, D6; A1,C6,D7;A1,C6,D8; A1,C6, D9; A1,C7, D1; A1,C7, D2; A1,C7,D3; A1,C7,D4; A1,C7,D5; A1,C7, D6; A1,C7,D7; A1,C7,D8; A1,C7, D9; A1,C8, D1; A1,C8, D2;A1,C8,D3; A1,C8,D4; A1,C8, D5; A1,C8, D6; A1,C8,D7; A1,C8,D8; A1,C8, D9;A1,C9, D1; A1,C9,D2; A1,C9,D3; A1,C9,D4; A1,C9, D5; A1,C9, D6; A1,C9,D7;A1,C9,D8; A1,C9, D9; A2,C1,D1; A2,C1,D2; A2,C1,D3; A2,C1,D4; A2,C1,D5;A2,C1,D6; A2,C1,D7; A2,C1,D8; A2,C1, D9; A2,C2,D1; A2,C2,D2; A2,C2,D3;A2,C2,D4; A2,C2,D5; A2,C2,D6; A2,C2,D7; A2,C2,D8; A2,C2, D9; A2,C3,D1;A2,C3,D2; A2,C3,D3; A2,C3,D4; A2,C3,D5; A2,C3,D6; A2,C3,D7; A2,C3,D8;A2,C3, D9; A2,C4, D1; A2,C4,D2; A2, C4,D3; A2,C4,D4; A2,C4, D5; A2,C4,D6; A2,C4,D7; A2, C4,D8; A2,C4, D9; A2,C5,D1; A2,C5,D2; A2,C5,D3;A2,C5,D4; A2,C5,D5; A2,C5,D6; A2,C5,D7; A2,C5,D8; A2,C5,D9; A2,C6,D1;A2,C6,D2; A2,C6,D3; A2,C6,D4; A2,C6,D5; A2,C6,D6; A2,C6,D7; A2,C6,D8;A2,C6,D9; A2,C7,D1; A2,C7,D2; A2,C7,D3; A2,C7,D4; A2,C7,D5; A2,C7,D6;A2,C7,D7; A2,C7,D8; A2,C7,D9; A2,C8,D1; A2,C8,D2; A2,C8,D3; A2,C8,D4;A2,C8,D5; A2,C8,D6; A2,C8,D7; A2,C8,D8; A2,C8,D9; A2,C9,D1; A2,C9,D2;A2,C9,D3; A2,C9,D4; A2,C9,D5; A2,C9,D6; A2,C9,D7; A2,C9,D8; A2,C9,D9;A3,C1,D1; A3,C1,D2; A3,C1,D3; A3,C1,D4; A3,C1,D5; A3,C1,D6; A3,C1,D7;A3,C1,D8; A3,C1,D9; A3,C2,D1; A3,C2,D2; A3,C2,D3; A3,C2,D4; A3,C2,D5;A3,C2,D6; A3,C2,D7; A3,C2,D8; A3,C2, D9; A3,C3,D1; A3,C3,D2; A3,C3,D3;A3,C3,D4; A3,C3,D5; A3,C3,D6; A3,C3,D7; A3,C3,D8; A3,C3,D9; A3,C4,D1;A3,C4,D2; A3,C4,D3; A3,C4,D4; A3,C4,D5; A3,C4,D6; A3,C4,D7; A3,C4,D8;A3,C4,D9; A3,C5,D1; A3,C5,D2; A3,C5,D3; A3,C5,D4; A3,C5,D5; A3,C5,D6;A3,C5,D7; A3,C5,D8; A3,C5,D9; A3,C6,D1; A3,C6,D2; A3,C6,D3; A3,C6,D4;A3,C6,D5; A3,C6,D6; A3,C6,D7; A3,C6,D8; A3,C6,D9; A3,C7,D1; A3,C7,D2;A3,C7,D3; A3,C7,D4; A3,C7,D5; A3,C7,D6; A3,C7,D7; A3,C7,D8; A3,C7,D9;A3,C8,D1; A3,C8,D2; A3,C8,D3; A3,C8,D4; A3,C8,D5; A3,C8,D6; A3,C8,D7;A3,C8,D8; A3,C8,D9; A3,C9,D1; A3,C9,D2; A3,C9,D3; A3,C9,D4; A3,C9,D5;A3,C9,D6; A3,C9,D7; A3,C9,D8; A3,C9,D9; A4,C1,D1; A4,C1,D2; A4,C1,D3;A4,C1,D4; A4,C1,D5; A4,C1,D6; A4,C1,D7; A4,C1,D8; A4,C1,D9; A4,C2,D1;A4,C2,D2; A4,C2,D3; A4,C2,D4; A4,C2,D5; A4,C2,D6; A4,C2,D7; A4,C2,D8;A4,C2,D9; A4,C3,D1; A4,C3,D2; A4,C3,D3; A4,C3,D4; A4,C3,D5; A4,C3,D6;A4,C3,D7; A4,C3,D8; A4,C3,D9; A4,C4,D1; A4,C4,D2; A4,C4,D3; A4,C4,D4;A4,C4,D5; A4,C4,D6; A4,C4,D7; A4,C4,D8; A4,C4,D9; A4,C5,D1; A4,C5,D2;A4,C5,D3; A4,C5,D4; A4,C5,D5; A4,C5,D6; A4,C5,D7; A4,C5,D8; A4,C5,D9;A4,C6,D1; A4,C6,D2; A4,C6,D3; A4,C6,D4; A4,C6,D5; A4,C6,D6; A4,C6,D7;A4,C6,D8; A4,C6,D9; A4,C7,D1; A4,C7,D2; A4,C7,D3; A4,C7,D4; A4,C7,D5;A4,C7,D6; A4,C7,D7; A4,C7,D8; A4,C7,D9; A4,C8,D1; A4,C8,D2; A4,C8,D3;A4,C8,D4; A4,C8,D5; A4,C8,D6; A4,C8,D7; A4,C8,D8; A4,C8,D9; A4,C9,D4;A4,C9,D2; A4,C9,D3; A4,C9,D4; A4,C9,D5; A4,C9,D6; A4,C9,D7; A4,C9,D8;A4,C9, D9; A5,C1,D1; A5,C1,D2; A5,C1,D3; A5,C1,D4; A5,C1,D5; A5,C1,D6;A5,C1,D7; A5,C1,D8; A5,C1,D9; A5,C2,D1; A5,C2,D2; A5,C2,D3; A5,C2,D4;A5,C2,D5; A5,C2,D6; A5,C2,D7; A5,C2,D8; A5,C2,D9; A5,C3,D1; A5,C3,D2;A5,C3,D3; A5,C3,D4; A5,C3,D5; A5,C3,D6; A5,C3,D7; A5,C3,D8; A5,C3,D9;A5,C4,D1; A5,C4,D2; A5,C4,D3; A5,C4,D4; A5,C4,D5; A5,C4,D6; A5,C4,D7;A5,C4,D8; A5,C4,D9; A5,C5,D1; A5,C5,D2; A5,C5,D3; A5,C5,D4; A5,C5,D5;A5,C5,D6; A5,C5,D7; A5,C5,D8; A5,C5,D9; A5,C6,D1; A5,C6,D2; A5,C6,D3;A5,C6,D4; A5,C6,D5; A5,C6,D6; A5,C6,D7; A5,C6,D8; A5,C6,D9; A5,C7,D1;A5,C7,D2; A5,C7,D3; A5,C7,D4; A5,C7,D5; A5,C7,D6; A5,C7,D7; A5,C7,D8;A5,C7,D9; A5,C8,D1; A5,C8,D2; A5,C8,D3; A5,C8,D4; A5,C8,D5; A5,C8,D6;A5,C8,D7; A5,C8,D8; A5,C8,D9; A5,C9,D1; A5,C9,D2; A5,C9,D3; A5,C9,D4;A5,C9,D5; A5,C9,D6; A5,C9,D7; A5,C9,D8; A5,C9,D9; A6,C1,D1; A6,C1,D2;A6,C1,D3; A6,C1,D4; A6,C1,D5; A6,C1,D6; A6,C1,D7; A6,C1,D8; A6,C1,D9;A6,C2,D1; A6,C2,D2; A6,C2,D3; A6,C2,D4; A6,C2,D5; A6,C2,D6; A6,C2,D7;A6,C2,D8; A6,C2,D9; A6,C3,D1; A6,C3,D2; A6,C3,D3; A6,C3,D4; A6,C3,D5;A6,C3, D6; A6,C3,D7; A6,C3,D8; A6,C3, D9; A6,C4, D1; A6,C4,D2; A6,C4,D3;A6,C4,D4; A6,C4, D5; A6,C4, D6; A6,C4,D7; A6,C4,D8; A6,C4, D9; A6,C5,D1;A6,C5,D2; A6,C5,D3; A6,C5,D4; A6,C5,D5; A6,C5,D6; A6,C5,D7; A6,C5,D8;A6,C5, D9; A6,C6,D1; A6,C6,D2; A6,C6,D3; A6,C6,D4; A6,C6,D5; A6,C6,D6;A6,C6,D7; A6,C6,D8; A6,C6, D9; A6,C7,D1; A6,C7,D2; A6,C7,D3; A6,C7,D4;A6,C7,D5; A6,C7,D6; A6,C7,D7; A6,C7,D8; A6,C7, D9; A6,C8,D1; A6,C8,D2;A6,C8,D3; A6,C8,D4; A6,C8,D5; A6,C8, D6; A6,C8,D7; A6,C8,D8; A6,C8, D9;A6,C9,D1; A6,C9,D2; A6,C9,D3; A6,C9,D4; A6,C9,D5; A6,C9,D6; A6,C9,D7;A6,C9,D8; A6,C9, D9; A1,C1,E1; A1,C1,E2; A1,C1,E3; A1,C2,E1; A1,C2,E2;A1,C2,E3; A1,C3,E1; A1,C3,E2; A1,C3,E3; A1,C4,E1; A1,C4,E2; A1,C4,E3;A1,C5,E1; A1,C5,E2; A1,C5,E3; A1,C6,E1; A1,C6,E2; A1,C6,E3; A1,C7,E1;A1,C7,E2; A1,C7,E3; A1,C8,E1; A1,C8,E2; A1,C8,E3; A1,C9, E1; A1,C9,E2;A1,C9,E3; A2,C1,E1; A2,C1,E2; A2,C1,E3; A2,C2,E1; A2,C2,E2; A2,C2,E3;A2,C3,E1; A2,C3,E2; A2,C3,E3; A2,C4,E1; A2,C4,E2; A2,C4,E3; A2,C5,E1;A2,C5,E2; A2,C5,E3; A2,C6,E1; A2,C6,E2; A2,C6,E3; A2,C7,E1; A2,C7,E2;A2,C7,E3; A2,C8,E1; A2,C8,E2; A2,C8,E3; A2,C9, E1; A2,C9,E2; A2,C9,E3;A3,C1,E1; A3,C1,E2; A3,C1,E3; A3,C2,E1; A3,C2,E2; A3,C2,E3; A3,C3,E1;A3,C3,E2; A3,C3,E3; A3,C4,E1; A3,C4,E2; A3,C4,E3; A3,C5,E1; A3,C5,E2;A3,C5,E3; A3,C6,E1; A3,C6,E2; A3,C6,E3; A3,C7,E1; A3,C7,E2; A3,C7,E3;A3,C8,E1; A3,C8,E2; A3,C8,E3; A3,C9,E1; A3,C9,E2; A3,C9,E3; A4,C1,E1;A4,C1,E2; A4,C1,E3; A4,C2,E1; A4,C2,E2; A4,C2,E3; A4,C3,E1; A4,C3,E2;A4,C3,E3; A4,C4,E1; A4,C4,E2; A4,C4,E3; A4,C5,E1; A4,C5,E2; A4,C5,E3;A4,C6,E1; A4,C6,E2; A4,C6,E3; A4,C7,E1; A4,C7,E2; A4,C7,E3; A4,C8,E1;A4,C8,E2; A4,C8,E3; A4,C9,E1; A4,C9,E2; A4,C9,E3; A5,C1,E1; A5,C1,E2;A5,C1,E3; A5,C2,E1; A5,C2,E2; A5,C2,E3; A5,C3, E1; A5,C3,E2; A5,C3,E3;A5,C4,E1; A5,C4,E2; A5,C4,E3; A5,C5,E1; A5,C5,E2; A5,C5,E3; A5,C6,E1;A5,C6,E2; A5,C6,E3; A5,C7,E1; A5,C7,E2; A5,C7,E3; A5,C8,E1; A5,C8,E2;A5,C8,E3; A5,C9,E1; A5,C9,E2; A5,C9,E3; A6,C1,E1; A6,C1,E2; A6,C1,E3;A6,C2,E1; A6,C2,E2; A6,C2,E3; A6,C3,E1; A6,C3,E2; A6,C3,E3; A6,C4,E1;A6,C4,E2; A6,C4,E3; A6,C5,E1; A6,C5,E2; A6,C5,E3; A6,C6,E1; A6,C6,E2;A6,C6,E3; A6,C7,E1; A6,C7,E2; A6,C7,E3; A6,C8,E1; A6,C8,E2; A6,C8,E3;A6,C9,E1; A6,C9,E2; A6,C9,E3; A1,D1,E1; A1,D1,E2; A1,D1,E3; A1,D2,E1;A1,D2,E2; A1,D2,E3; A1,D3,E1; A1,D3,E2; A1,D3,E3; A1,D4,E1; A1,D4,E2;A1,D4,E3; A1,D5,E1; A1,D5,E2; A1,D5,E3; A1,D6,E1; A1,D6,E2; A1,D6,E3;A1,D7,E1; A1,D7,E2; A1,D7,E3; A1,D8,E1; A1,D8,E2; A1,D8,E3; A1,D9,E1;A1,D9,E2; A1,D9,E3; A2,D1,E1; A2,D1,E2; A2,D1,E3; A2,D2,E1; A2,D2,E2;A2,D2,E3; A2,D3,E1; A2,D3,E2; A2,D3,E3; A2,D4,E1; A2,D4,E2; A2,D4,E3;A2,D5,E1; A2,D5,E2; A2,D5,E3; A2,D6,E1; A2,D6,E2; A2,D6,E3; A2,D7,E1;A2,D7,E2; A2,D7,E3; A2,D8,E1; A2,D8,E2; A2,D8,E3; A2,D9,E1; A2,D9,E2;A2,D9,E3; A3,D1,E1; A3,D1,E2; A3,D1,E3; A3,D2,E1; A3,D2,E2; A3,D2,E3;A3,D3,E1; A3,D3,E2; A3,D3,E3; A3,D4,E1; A3,D4,E2; A3,D4,E3; A3,D5,E1;A3,D5,E2; A3,D5,E3; A3,D6,E1; A3,D6,E2; A3,D6,E3; A3,D7,E1; A3,D7,E2;A3,D7,E3; A3,D8,E1; A3,D8,E2; A3,D8,E3; A3,D9,E1; A3,D9,E2; A3,D9,E3;A4,D1,E1; A4,D1,E2; A4,D1,E3; A4,D2,E1; A4,D2,E2; A4,D2,E3; A4,D3,E1;A4,D3,E2; A4,D3,E3; A4,D4,E1; A4,D4,E2; A4,D4,E3; A4,D5,E1; A4,D5,E2;A4,D5,E3; A4,D6,E1; A4,D6,E2; A4,D6,E3; A4, D7, E1; A4, D7, E2; A4, D7,E3; A4,D8,E1; A4,D8,E2; A4,D8,E3; A4,D9,E1; A4,D9,E2; A4,D9,E3;A5,D1,E1; A5,D1,E2; A5,D1,E3; A5,D2,E1; A5,D2,E2; A5,D2,E3; A5,D3,E1;A5,D3,E2; A5,D3,E3; A5,D4,E1; A5,D4,E2; A5,D4,E3; A5,D5,E1; A5,D5,E2;A5,D5,E3; A5,D6,E1; A5,D6,E2; A5,D6,E3; A5, D7, E1; A5, D7, E2; A5, D7,E3; A5,D8,E1; A5,D8,E2; A5,D8,E3; A5,D9,E1; A5,D9,E2; A5,D9,E3;A6,D1,E1; A6,D1,E2; A6,D1,E3; A6,D2,E1; A6,D2,E2; A6,D2,E3; A6,D3,E1;A6,D3,E2; A6,D3,E3; A6,D4,E1; A6,D4,E2; A6,D4,E3; A6,D5,E1; A6,D5,E2;A6,D5,E3; A6,D6,E1; A6,D6,E2; A6,D6,E3; A6,D7,E1; A6, D7, E2; A6, D7,E3; A6,D8,E1; A6,D8,E2; A6,D8,E3; A6,D9,E1; A6,D9,E2; A6,D9,E3;B1,C1,D1; B1,C1,D2; B1,C1,D3; B1,C1,D4; B1,C1,D5; B1,C1,D6; B1,C1,D7;B1,C1,D8; B1,C1,D9; B1,C2,D1; B1,C2; B1,C3; B1,C2,D4; B1,C2,D5;B1,C2,D6; B1,C2,D7; B1,C2,D8; B1,C2,D9; B1,C3,D1; B1,C3,D2; B1,C3,D3;A1,C3,D4; B1,C3,D5; B1,C3,D6; B1,C3,D7; B1,C3,D8; B1,C3,D9; B1,C4,D1;B1,C4,D2; B1,C4,D3; B1,C4,D4; B1,C4,D5; B1,C4,D6; B1,C4,D7; B1,C4,D8;B1,C4,D9; B1,C5,D1; B1,C5,D2; B1,C5,D3; B1,C5,D4; B1,C5,D5; B1,C5,D6;B1,C5,D7; B1,C5,D8; B1,C5,D9; B1,C6,D1; B1,C6,D2; B1,C6,D3; B1,C6,D4;B1,C6,D5; B1,C6,D6; B1,C6,D7; B1,C6,D8; B1,C6,D9; B1,C7,D1; B1,C7,D2;B1,C7,D3; B1,C7,D4; B1,C7,D5; B1,C7,D6; B1,C7,D7; B1,C7,D8; B1,C7,D9;B1,C8,D1; B1,C8,D2; B1,C8,D3; B1,C8,D4; B1,C8,D5; B1,C8,D6; B1,C8,D7;B1,C8,D8; B1,C8,D9; B1,C9,D1; B1,C9,D2; B1,C9,D3; B1,C9,D4; B1,C9,D5;B1,C9,D6; B1,C9,D7; B1,C9,D8; B1,C9,D9; B2,C1,D1; B2,C1,D2; B2,C1,D3;B2,C1,D4; B2,C1,D5; B2,C1,D6; B2,C1,D7; B2,C1,D8; B2,C1,D9; B2,C2,D1;B2,C2,D2; B2,C2,D3; B2,C2,D4; B2,C2,D5; B2,C2,D6; B2,C2,D7; B2,C2,D8;B2,C2,D9; B2,C3,D1; B2,C3,D2; B2,C3,D3; B2,C3,D4; B2,C3,D5; B2,C3,D6;B2,C3,D7; B2,C3,D8; B2,C3,D9; B2,C21,D1; B2,C4,D2; B2,C4,D3; B2,C14,D4;B2,C4,D5; B2,C4,D6; B2,C4,D7; B2,C4,D8; B2,C4, D9; B2,C5,D1; B2,C5,D2;B2,C5,D3; B2,C5,D4; B2,C5,D5; B2,C5,D6; B2,C5,D7; B2,C5,D8; B2,C5, D9;B2,C6,D1; B2,C6,D2; B2,C6,D3; B2,C6,D4; B2,C6,D5; B2,C6,D6; B2,C6,D7;B2,C6,D8; B2,C6,D9; B2,C7,D1; B2,C7,D2; B2,C7,D3; B2,C7,D4; B2,C7,D5;B2,C7,D6; B2,C7,D7; B2,C7,D8; B2,C7,D9; B2,C8,D1; B2,C8,D2; B2,C8,D3;B2,C8,D5; B2,C8,D6; B2,C8,D7; B2,C8,D8; B2,C8, D9; B2,C9,D1; B2,C9,D2;B2,C9,D3; B2,C9,D4; B2,C9,D5; B2,C9,D6; B2,C9,D7; B2,C9,D8; B2,C9,D9;B3,C1,D1; B3,C1,D2; B3,C1,D3; B3,C1,D4; B3,C1,D5; B3,C1,D6; B3,C1,D7;B3,C1,D8; B3,C1,D9; B3,C2,D1; B3,C2,D2; 83,C2,D3; B3,C2,D4; B3,C2,D5;B3,C2,D6; B3,C2,D7; B3,C2,D8; B3,C2,D9; B3,C3,D1; B3,C3,D2; B3,C3,D3;B3,C3,D4; B3,C3,D5; B3,C3,D6; B3,C3,D7; B3,C3,D8; B3,C3,D9; B3,C4,D1;B3,C4,D2; B3,C4,D3; B3,C4,D4; B3,C4,D5; B3,C4,D6; B3,C4,D7; B3,C4,D8;B3,C4, D9; B3,C5,D1; B3,C5,D2; B3,C5,D3; B3,C5,D4; B3,C5,D5; B3,C5,D6;B3,C5,D7; B3,C5,D8; B3,C5,D9; B3,C6,D1; B3,C6,D2; B3,C6,D3; B3,C6,D4;B3,C6,D5; B3,C6,D6; B3,C6,D7; B3,C6,D8; B3,C6,D9; B3,C7,D1; B3,C7,D2;B3,C7,D3; B3,C7,D4; B3,C7,D5; B3,C7,D6; B3,C7,D7; B3,C7,D8; B3,C7,D9;B3,C8,D1; B3,C8,D2; B3,C8,D3; B3,C8,D4; B3,C8,D5; B3,C8,D6; B3,C8,D7;B3,C8,D8; B3,C8, D9; B3,C9,D1; B3,C9,D2; B3,C9,D3; B3,C9,D4; B3,C9,D5;B3,C9,D6; B3,C9,D7; B3,C9,D8; B3,C9,D9; B4,C1,D1; B4,C1,D2; B4,C1,D3;B4,C1,D4; B4,C1,D5; B4,C1,D6; B4,C1,D7; B4,C1,D8; B4,C1,D9; B4,C2,D1;B4,C2,D2; B4,C2,D3; B4,C2,D4; B4,C2,D5; B4,C2,D6; B4,C2,D7; B4,C2,D8;B4,C2,D9; B4,C3,D1; B4,C3,D2; B4,C3,D3; B4,C3,D4; B4,C3,D5; B4,C3,D6;B4,C3,D7; B4,C3,D8; B4,C3,D9; B4,C4,D1; B4,C4,D2; B4,C4,D3; B4,C4,D4;B4,C4,D5; B4,C4,D6; B4,C4,D7; B4,C4,D8; B4,C4,D9; B4,C5,D1; B4,C5,D2;B4,C5,D3; B4,C5,D4; B4,C5,D5; B4,C5,D6; B4,C5,D7; B4,C5,D8; B4,C5,D9;B4,C6,D1; B4,C6,D2; B4,C6,D3; B4,C6,D4; B4,C6,D5; B4,C6,D6; B4,C6,D7;B4,C6,D8; B4,C6,D9; B4,C7,D1; B4,C7,D2; B4,C7,D3; B4,C7,D4; B4,C7,D5;B4,C7,D6; B4,C7,D7; B4,C7,D8; B4,C7,D9; B4,C8,D1; B4,C8,D2; B4,C8,D3;B4,C8,D4; B4,C8,D5; B4,C8,D6; B4,C8,D7; B4,C8,D8; B4,C8,D9; B4,C9,D1;B4,C9,D2; B4,C9,D3; B4,C9,D4; B4,C9,D5; B4,C9,D6; B4,C9,D7; B4,C9,D8;B4,C9,D9; B1,C1,E1; B1,C1,E2; B1,C1,E3; B1,C2,E1; B1,C2,E2; B1,C2,E3;B1,C3,E1; B1,C3,E2; B1,C3,E3; B1,C4,E1; B1,C4,E2; B1,C4,E3; B1,C5,E1;B1,C5,E2; B1,C5,E3; B1,C6,E1; B1,C6,E2; B1,C6,E3; B1,C7,E1; B1,C7,E2;B1,C7,E3; B1,C8,E1; B1,C8,E2; B1,C8,E3; B1,C9,E1; B1,C9,E2; B1,C9,E3;B2,C1,E1; B2,C1,E2; B2,C1,E3; B2,C2,E1; B2,C2,E2; B2,C2,E3; B2,C3,E1;B2,C3,E2; B2,C3,E3; B2,C4,E1; B2,C4,E2; B2,C4,E3; B2,C5,E1; B2,C5,E2;B2,C5,E3; B2,C6,E1; B2,C6,E2; B2,C6,E3; B2,C7,E1; B2,C7,E2; B2,C7,E3;B2,C8,E1; B2,C8,E2; B2,C8,E3; B2,C9,E1; B2,C9,E2; B2,C9,E3; B3,C1,E1;B3,C1,E2; B3,C1,E3; B3,C2,E1; B3,C2,E2; B3,C2,E3; B3,C3,E1; B3,C3,E2;B3,C3,E3; B3,C4,E1; B3,C4,E2; B3,C4,E3; B3,C5,E1; B3,C5,E2; B3,C5,E3;B3,C6,E1; B3,C6,E2; B3,C6,E3; B3,C7,E1; B3,C7,E2; B3,C7,E3; B3,C8,E1;B3,C8,E2; B3,C8,E3; B3,C9,E1; B3,C9,E2; B3,C9,E3; B4,C1,E1; B4,C1,E2;B4,C1,E3; B4,C2,E1; B4,C2,E2; B4,C2,E3; B4,C3,E1; B4,C3,E2; B4,C3,E3;B4,C4,E1; B4,C4,E2; B4,C4,E3; B4,C5,E1; B4,C5,E2; B4,C5,E3; B4,C6,E1;B4,C6,E2; B4,C6,E3; B4,C7,E1; B4,C7,E2; B4,C7,E3; B4,C8,E1; B4,C8,E2;B4,C8,E3; B4,C9,E1; B4,C9,E2; B4,C9,E3; B1,D1,E1; B1,D1,E2; B1,D1,E3;B1,D2,E1; B1,D2,E2; B1,D2,E3; B1,D3,E1; B1,D3,E2; B1,D3,E3; B1,D4,E1;B1,D4,E2; B1,D4,E3; B1,D5,E1; B1,D5,E2; B1,D5,E3; B1,D6,E1; B1,D6,E2;B1,D6,E3; B1,D7,E1; B1,D7,E2; B1,D7,E3; B1,D8,E1; B1,D8,E2; B1,D8,E3;B1,D9,E1; B1,D9,E2; B1,D9,E3; B2,D1,E1; B2,D1,E2; B2,D1,E3; B2,D2,E1;B2,D2,E2; B2,D2,E3; B2,D3,E1; B2,D3,E2; B2,D3,E3; B2,D4,E1; B2,D4,E2;B2,D4,E3; B2,D5,E1; B2,D5,E2; B2,D5,E3; B2,D6,E1; B2,D6,E2; B2,D6,E3;B2,D7,E1; B2,D7,E2; B2,D7,E3; B2,D8,E1; B2,D8,E2; B2,D8,E3; B2,D9,E1;B2,D9,E2; B2,D9,E3; B3,D1,E1; B3,D1,E2; B3,D1,E3; B3,D2,E1; B3,D2,E2;B3,D2,E3; B3,D3,E1; B3,D3,E2; B3,D3,E3; B3,D4,E1; B3,D4,E2; B3,D4,E3;B3,D5,E1; B3,D5,E2; B3,D5,E3; B3,D6,E1; B3,D6,E2; B3,D6,E3; B3,D7, E1;B3,D7,E2; B3, D7,E3; B3,D8,E1; B3,D8,E2; B3,D8,E3; B3,D9,E1; B3,D9,E2;B3,D9,E3; B4,D1,E1; B4,D1,E2; B4,D1,E3; B4,D2,E1; B4,D2,E2; B4,D2,E3;B4,D3,E1; B4,D3,E2; B4,D3,E3; B4,D4,E1; B4,D4,E2; B4,D4,E3; B4,D5,E1;B4,D5,E2; B4,D5,E3; B4,D6,E1; B4,D6,E2; B4,D6,E3; B4,D7,E1; B4,D7,E2;B4,D7,E3; B4,D8,E1; B4,D8,E2; B4,D8,E3; B4,D9,E1; B4,D9,E2; B4,D9,E3;C1,D1,E1; C1,D1,E2; C1,D1,E3; C1,D2,E1; C1,D2,E2; C1,D2,E3; C1,D3,E1;C1,D3,E2; C1,D3,E3; C1,D4,E1; C1,D4,E2; C1,D4,E3; C1,D5,E1; C1,D5,E2;C1,D5,E3; C1,D6,E1; C1,D6,E2; C1,D6,E3; C1,D7,E1; C1,D7,E2; C1,D7,E3;C1,D8,E1; C1,D8,E2; C1,D8,E3; C1,D9,E1; C1,D9,E2; C1,D9,E3; C2,D1,E1;C2,D1,E2; C2,D1,E3; C2,D2,E1; C2,D2,E2; C2,D2,E3; C2,D3,E1; C2,D3,E2;C2,D3,E3; C2,D4,E1; C2,D4,E2; C2,D4,E3; C2,D5,E1; C2,D5,E2; C2,D5,E3;C2,D6,E1; C2,D6,E2; C2,D6,E3; C2,D7,E1; C2,D7,E2; C2,D7,E3; C2,D8,E1;C2,D8,E2; C2,D8,E3; C2,D9,E1; C2,D9,E2; C2,D9,E3; C3,D1,E1; C3,D1,E2;C3,D1,E3; C3,D2,E1; C3,D2,E2; C3,D2,E3; C3,D3,E1; C3,D3,E2; C3,D3,E3;C3,D4,E1; C3,D4,E2; C3,D4,E3; C3,D5,E1; C3,D5,E2; C3,D5,E3; C3,D6,E1;C3,D6,E2; C3,D6,E3; C3,D7,E1; C3,D7,E2; C3,D7,E3; C3,D8,E1; C3,D8,E2;C3,D8,E3; C3,D9,E1; C3,D9,E2; C3,D9,E3; C4,D1,E1; C4,D1,E2; C4,D1,E3;C4,D2,E1; C4,D2,E2; C4,D2,E3; C4,D3,E1; C4,D3,E2; C4,D3,E3; C4,D4,E1;C4,D4,E2; C4,D4,E3; C4,D5,E1; C4,D5,E2; C4,D5,E3; C4,D6,E1; C4,D6,E2;C4,D6,E3; C4,D7,E1; C4,D7,E2; C4,D7,E3; C4,D8,E1; C4,D8,E2; C4,D8,E3;C4,D9,E1; C4,D9,E2; C4,D9,E3; C5,D1,E1; C5,D1,E2; C5,D1,E3; C5,D2,E1;C5,D2,E2; C5,D2,E3; C5,D3,E1; C5,D3,E2; C5,D3,E3; C5,D4,E1; C5,D4,E2;C5,D4,E3; C5,D5,E1; C5,D5,E2; C5,D5,E3; C5,D6,E1; C5,D6,E2; C5,D6,E3;C5,D7,E1; C5,D7,E2; C5,D7,E3; C5,D8,E1; C5,D8,E2; C5,D8,E3; C5,D9,E1;C5,D9,E2; C5,D9,E3; C6,D1,E1; C6,D1,E2; C6,D1,E3; C6,D2,E1; C6,D2,E 2;C6,D2,E3; C6,D3,E1; C6,D3,E2; C6,D3,E3; C6,D4,E1; C6,D4,E2; C6,D4,E3;C6,D5,E1; C6,D5,E2; C6,D5,E3; C6,D6,E1; C6,D6,E2; C6,D6,E3; C6,D7,E1;C6, D7,E2; C6,D7,E3; C6,D8,E1; C6,D8,E2; C6,D8,E3; C6,D9,E1; C6,D9,E2;C6,D9,E3; C7,D1, E1; C7,D1,E2; C7,D1,E3; C7,D2,E1; C7,D2,E2; C7,D2,E3;C7,D3,E1; C7,D3,E2; C7,D3,E3; C7,D4,E1; C7,D4,E2; C7,D4,E3; C7,D5,E1;C7,D5,E2; C7,D5,E3; C7,D6,E1; C7,D6,E2; C7,D6,E3; C7,D7,E1; C7,D7,E2;C7,D7,E3; C7,D8,E1; C7,D8,E2; C7,D8,E3; C7,D9,E1; C7,D9,E2; C7,D9,E3;C8,D1,E1; C8,D1,E2; C8,D1,E3; C8,D2,E1; C8,D2,E2; C8,D2,E3; C8,D3,E1;C8,D3,E2; C8,D3,E3; C8,D4,E1; C8,D4,E2; C8,D4,E3; C8,D5,E1; C8,D5,E2;C8,D5,E3; C8,D6,E1; C8,D6,E2; C8,D6,E3; C8,D7,E1; C8,D7,E2; C8,D7,E3;C8,D8,E1; C8,D8,E2; C8,D8,E3; C8,D9,E1; C8,D9,E2; C8,D9,E3; C9,D1,E1;C9,D1,E2; C9,D1,E3; C9,D2,E1; C9,D2,E2; C9,D2,E3; C9,D3,E1; C9,D3,E2;C9,D3,E3; C9,D4,E1; C9,D4,E2; C9,D4, E3; C9,D5,E1; C9,D5,E2; C9,D5,E3;C9,D6,E1; C9,D6,E2; C9,D6,E3; C9,D7,E1; C9,D7,E2; C9,D7, E3; C9,D8,E1;C9,D8,E2; C9,D8,E3; C9,D9,E1; C9,D9,E2; C9,D9,E3;

Each of these combinations shows an overadditive effect in the treatmentof inflammatory diseases especially respiratory diseases of inflammatoryorigin as asthma or COPD, or also rheumatism and auto-immune diseases.

Each of the combinations may be administered as fixed of freecombinations, which may be administered simultaneously or one after theother.

Each of the combinations may be administered orally, topically,preferably inhalative. In free combinations the single active substancesmay be presented in the same or different administration forms, chosenfrom the possibilities of oral, topical, and inhalative application.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the influence of glycopyrrolte, rolipram and budesonide andtheir combinations on the LPS stimulated TNF-α release from humanmonocytes. The y-axis indicates the inhibition of TNF-α release in %.The size of the columns shows the combination of substances and theiramounts applied in the experiment. Statistically significantover-additive inhibition is indicated above the columns (p values).

FIG. 2 shows the influence of glycopyrrolate, formoterol and budesonideand their combinations on the LPS stimulated TNF-α release from humanmonocytes. The y-axis indicates the inhibition of TNF-α release in %.The size of the columns shows the inhibition of TNF-α release for eachcombination of substances. The table below the columns shows thecombination of substances and their amounts applied in the experiment.Statistically significant over-additive inhibition is indicated abovethe columns (p values).

EXPERIMENTAL PART

The influence of monocompounds and their various combinations, also inthree-in-one on TNF secretion was investigated by using human monocytes.The study was approved by our institutional Ethics Committee accordingto the International Declarations of Helsinki and Tokyo.

Peripheral blood mononuclear cells (PBMCs) were isolated fromheparinized blood samples of healthy donors by density gradientcentrifugation. An equal volume of Hanks buffer (Invitrogen, Heidelberg,Germany) was added to heparinized whole blood samples. 15 mlHistopaque-1077 (Sigma, Deisenhofen, Germany) were overlayed with amaximum of 40 ml of blood/Hanks mixture and were centrifuged for 30 minat room temperature (2000 rpm). A visible band containing PBMCs wastransferred to a fresh tube and washed twice with Hanks-buffer. Finallycells are seeded in RPMI 1640 Medium (Life Technologies, Heidelberg,Germany) with Glutamax I (Gibco BRL, Eggenstein) and 10% FCS (BoehringerMannheim, Penzberg, Germany). After isolated, PBMCs were seeded in RPMI1640 medium (Invitrogen, Heidelberg, Germany) with Glutamax I(Invitrogen, Heidelberg, Germany). PBMCs were cultured in RPMI 1640medium at 37° C. 5% CO₂ overnight. Monocytes were isolated from othercells by adherence method, non-adherent cells were removed by changingthe medium.

Cells were re-suspended at 10⁶ cells/ml and incubated in 500 μl volumesin 24-well tissue culture plates (Falcon Becton Dickinson Labware,Heidelberg, Germany) at 37° C., 5% CO₂. After pre-incubation with testsubstances (0.5 μl/500 μl medium) for 30 min, cells were stimulated withlipopolysaccharide (LPS) (Sigma, Deisenhofen, Germany) (1 μg/ml). After24 h of incubation, cells were sedimented by centrifugation. Thesupernatants were harvested and kept frozen at −80° C. until proteindetermination.

Cytokine measurements in culture supernatants were done by sandwichELISA using matched antibody pairs (BD Pharmingen, Heidelberg, Germany).ELISA plates (Maxisorb, Nunc, Wiesbaden, Germany) were coated overnightwith anti-cytokine monoclonal antibody (mAb) in 0.1 M carbonate buffer,pH 9.5. After being washed, plates were blocked with Assay Diluent (BDPharmingen, Heidelberg, Germany) for 1 h and washed again. Appropriatelydiluted supernatant samples and standards were distributed in duplicatesand the plates were incubated for 2 h at room temperature. Plates werewashed, incubated for 1 h with working detector (biotinylatedanti-cytokine antibody and Avidin-horseradish peroxidase conjugate) (BDPharmingen, Heidelberg, Germany). After washing, substrate (TMB andhydrogen peroxide) was added. The reaction was stopped by adding of 1MH₃PO₄. Plates were read at 450 nm (reference 570 nm) in a microplatereader (Dynatech, Alexandria, USA). The results were expressed as apercentage of the control level of cytokines production by cellsstimulated in the absence of the compound.

Upon LPS-stimulation, basal TNFα release from monocytes increased from328 pg/ml up to 7,258 pg/ml. R,R-glycopyrrolate alone did not influencethe LPS-induced TNFα release up to 10 μmol/l.

The PDE4 inhibitor rolipram dose dependently inhibited the TNFα release.Maximum inhibition was around 70%. Consequently, instead of IC₅₀, theIC₃₅-value was determined. The IC₃₅ for rolipram amounted to 68.9±15.2nM.

The corticosteroid budesonide also inhibited the TNFα release in adose-dependent manner. The IC₅₀ for budesonide was 0.55±0.13 nM.

The β₂-adrenoceptor agonists formoterol in concentrations of up to 10 μMdid not affect the LPS stimulated TNF-α release

Based on the results from the single substances, in a first experimentthe effect of the PDE4 inhibitor rolipram (10 nM) and the corticosteroidbudesonide (0.1 nM) alone and in combination, and in combination withR,R-glycopyrrolate (10 μM) on the LPS-induced TNFα release from humanPBMCs was investigated. The concentrations chosen for rolipram andbudesonide were considerably below their IC₃₅ and IC₅₀, respectively.The results are summarized in FIG. 1.

The PDE4 inhibitor rolipram dose dependently inhibited the TNFα release.Maximum inhibition was around 70%. Consequently, instead of IC₅₀, theIC₃₅-value was determined. The IC₃₅ for rolipram amounted to 68.9±15.2nM.

The corticosteroid budesonide also inhibited the TNFα release in adose-dependent manner. The IC₅₀ for budesonide was 0.55±0.13 nM.

The β₂-adrenoceptor agonists formoterol in concentrations of up to 10 μMdid not affect the LPS stimulated TNF-α release

Based on the results from the single substances, in a first experimentthe effect of the PDE4 inhibitor rolipram (10 nM) and the corticosteroidbudesonide (0.1 nM) alone and in combination, and in combination withR,R-glycopyrrolate (10 μM) on the LPS-induced TNFα release from humanPBMCs was investigated. The concentrations chosen for rolipram andbudesonide were considerably below their IC₃₅ and IC₅₀, respectively.The results are summarized in FIG. 1.

As can be seen from FIG. 1, each drug alone hardly affected LPS-inducedTNFα release. At the intentionally low concentrations, each doublecombination caused only a minor inhibition of TNFα secretion from PBMCs.In contrast, the three-in-one combination (budesonide, rolipram andR,R-glycopyrrolate) resulted in statistically significant over-additiveinhibition of the TNFα release.

Similar results were seen for the three-in-one combination (formoterol,budesonide and R,R-glycopyrrolate) which were evaluated in a secondexperiment. The double combination of R,R-glycopyrrolate/formoterol andR,R-glycopyrrolate/budesonide caused only a minor inhibition of TNF-αsecretion. The double combination of formoterol/budesonide inhibitedTNF-α secretion by about 25%. In contrast, the three-in-one combinationof formoterol, budesonide and R,R-glycopyrrolate most effectivelyinhibited TNF-α secretion by about 50%. This over-additive effect wasstatistically significant. The results are summarized in FIG. 2.

The medications can be administered in different ways, such asmetered-dosage inhalers (MDIs), in dry powder inhalers (DPIs), and inanother liquid formulation suitable for inhalation. They can also beadministered together in a single dosage form. Or they may beadministered in different dosage forms. They may be administered at thesame time. Or they may be administered either close in time or remotely,such as where one is administered in the morning and the second isadministered in the evening. The combination may be usedprophylactically or after the onset of symptoms has occurred. In someinstances the combination(s) may be used to prevent the progression of apulmonary disease or to arrest the decline of a function such as lungfunction.

These drugs, the anticholinergics, β₂-adrenoceptor agonist, the PDE4inhibitors and GCs, are usually administered as an aerosol, or as aninhaled powder. Presently available LTRAs are administered orally.However, there is convincing evidence that LTRAs are also effective whenthey are given topically. Zafirlukast administered topically into theeyes effectively inhibit the development of symptoms and mediatorrelease in an experimental model of allergic conjunctivitis in rats(Papathanassiou et al. Inflamm Res 2004; 53:373-6) indicating that theycould also be administered as an aerosol or powder. This inventioncontemplates either co-administering all drugs in one delivery form suchas an inhaler that is putting all drugs in the same inhaler.

As inhalable compositions pressurized metered dose inhalers, dry powdersor inhalation solutions without propellant can be considered. Among thelatter are even sterile, ready for use or just before use manufacturedinhalation solutions, suspensions or concentrates as a nebulizablecomposition in an aqueous and/or organic medium. These dosage forms arepart of the present invention.

Pressurized metered dose inhalers with propellants may contain theactive substances in solution or in dispersion in a propellant. Thepropellants which can be used for inhalation aerosols in this inventionare well known: mainly halogenated hydrocarbon derivatives, TG134a andTG227, or their mixtures are applied. Furthermore detergents (eg. oleicacid), stabilizers (eg. sodium edetate), co-solvents (eg.propyleneglycol, polyethyleneglycol, glycerol), antioxidants (eg.ascorbic acid), lubricants (eg. polyoxyethylene-glyceryl-trioleate) orbuffer systems or other excipients for pH adjustment (eg. hydrochloricacid) are normally added. The active ingredient may have an averageparticle diameter of up to 5 μm.

A combination of ethyl alcohol and polyoxyethylene-25-glyceryl-trioleate(trade name: Tagat TO) can be suitable used as adetergent/stabilizer/co-solvent/lubricant complex in a concentrationbetween 0.5 and 1.5%.

The above mentioned aerosols containing a propellant, solutions orsuspensions according to the invention are administered by state of theart inhalers, so-called pressurized metered dose inhalers (=pMDI). Theycan be equipped with different-sized metal or plastic stems responsiblefor metering and release of the actives.

Administration of the medicament or pharmaceutical composition ispreferably by inhalation. The inhalable form of the medicament may be,for example, an atomizable composition such as an aerosol comprising theactive ingredients, separately or in admixture, in solution ordispersion in a propellant, or a nebulizable composition comprising adispersion of the active ingredient in an aqueous/organic or medium. Forexample, the inhalable form of the medicament may be an aerosolcomprising a mixture of any composition according to the invention insolution or dispersion in a propellant, or a combination of an aerosolcontaining each single active substance in solution or dispersion in apropellant. In another example, the inhalable form is a nebulizedcomposition comprising a dispersion of the substances according to theinventive combination in an aqueous or organic medium, or a combinationof dispersions of each substance in such a medium.

In another embodiment of the invention, the inhalable form is a drypowder, i.e. the substances are present in a dry powder comprisingfinely divided each substance optionally together with a finely dividedpharmaceutically acceptable carrier, which is preferably present and maybe chosen from materials known as carriers in dry powder inhalationcompositions, for example saccharides, including monosaccharides,disaccharides, polysaccharides and sugar alcohols such as arabinose,fructose, ribose, mannose, sucrose, trehalose, lactose, starches,dextran or mannitol. An especially preferred carrier is lactose. The drypowder may be in capsules of gelatin or plastic, or in blisters, for ina dry powder inhalation device. Alternatively, the dry powder may becontained as a reservoir in a dose dry powder inhalation device.

The inhalation powders according to this invention can be administeredby the help of state of the art dry powder inhalation devices forinstance the Novolizer®. The inhalation powder can be pre-metered incapsules (eg. gelatine) or blisters (aluminium pouches) or metered justbefore use from a bulk reservoir. The active substances of the inventivecombination can be there in a fixed combination or both actives are inseparate packaging units, that they can be administered independent ofeach other from one device or a pack from two or more different devicesor simultaneously.

In the finely divided particulate form of the medicament, and in theaerosol composition where the active ingredient is present inparticulate form, the active ingredient may have an average particlediameter of up to 4 μm. The finely divided carrier, where present,generally has a maximum diameter up to approximately 500 μM andconveniently has a mean particle diameter of 10 to 350 μm, preferablyapprox. 110 to 290 μm. The particle size of the active ingredient, andthat of the carrier where present in dry powder compositions, can bereduced to the desired level by conventional methods, for example bygrinding in an air-jet mill, ball mill or vibrator mill,microprecipitation, spray-drying, lyophilisation or recrystallisationfrom supercritical media.javascriptpopup(‘citart1’,‘b117b120b152b153b154’,‘10.1111/j.1368-5031.2005.00509.x’,‘bj13685031200500509x’,”)

The active ingredients may be given from 1 to 8 times a day, sufficientto exhibit the desired activity. Preferably, the active components aregiven about once or four times a day, more preferably once or twice aday.

The inhaled anticholinergic drug, can be administered in an amount ofbetween 5 and 500 μg/day adult human with the preference of 15 to 300μg/day.

β2-adrenoceptor agonists can be administered in different amountsdependent on substance used, for example for formoterol nominal doses of1 to 20 μg, for salmeterol 10 to 200 μg.

The PDE4 inhibitor can be administered in an amount between 10 and 5000μg/day adult human with the preference of 50 to 2000 μg/day independence of the intensity of the airway inflammation.

The glucocorticoid can be administered in an amount of between 50 and2000 μg/day adult human with the preference of 100 to 1000 mg/day independence of the intensity of the airway inflammation.

The LT-receptor antagonist can be administered in an amount of between 1and 1000 μg/day adult human with the preference of 1 to 500 μg/dayespecially preferred 1 to 100 mg/day.

The active ingredients in all above aerosol formulations are preferablyin the concentration of 0.01 wt % to 5 wt % of the total formulation.

For the treatment of Autoimmune diseases the drugs can preferably beadministered either by oral route or rectally as enema.

The anticholinergic drug, R,R-glycopyrrolate can be administered in anamount between 1 and 199 mg/day for adult patients with the preferenceof 5 to 50 mg/day.

Basically, the initial doses of oral corticosteroids (for exampleprednisolone) may vary from 5 mg to 60 mg per day depending on thespecific disease entity being treated. In situations of less severitylower doses will generally suffice while in selected patients higherinitial doses may be required. The recommended dosage of budesonide is6-9 mg daily.

The usual dosage of PDE4 inhibitors are different: for example forcilomilast, it is at 30 mg/day or for roflumilast, it varies between0.25-1 mg/day.

The dosages of LTRAs vary over a great band width: For example the usualdaily dose of montelukast amounts to 10 mg. Similarly, the daily dose ofzafirlukast is 20 mg twice daily. However, the dose of pranlukast is 225mg twice daily.

It is contemplated that all active agents would be administered at thesame time, or very close in time. Alternatively, one drug could be takenin the morning and others later in the day. Or in another scenario, onedrug could be taken twice daily and the others once daily, either at thesame time as one of the twice-a-day dosing occurred, or separately.Preferably all drugs would be taken together at the same time.

1. A combination for the treatment of inflammatory diseases comprisingat least three different pharmaceutically active substances or theirphysiologically acceptable salts selected from the group consisting ofanticholinergics, glucocorticoids, and β₂-adrenoceptor agonists.
 2. Thecombination according to claim 1, wherein the anticholinergic is racemicglycopyrrolate, an enantiomer of glycopyrrolate, a diastereoisomer ofglycopyrrolate, physiologically acceptable salts thereof or a mixturethereof.
 3. The combination according to claim 1, wherein theglucocorticoid is selected from the group consisting of budesonide,fluticasone, mometasone, beclomethasone, ciclesonide, triamcinolone,loteprednol, etiprednol, flunisolide and physiologically acceptablesalts thereof.
 4. The combination according to claim 1, wherein theβ₂-adrenoceptor agonist is selected from the group consisting ofsalbutamol, terbutaline, salmeterol, formoterol, indacaterol, fenoterol,reproterol, pirbuterol, bitolterol and physiologically acceptable saltsthereof.
 5. The combination according to claim 1, wherein theinflammatory disease is selected from the group consisting of arespiratory disease selected from asthma and COPD; and rheumatism or anauto-immune disease selected from rheumatoid arthritis,glomerulonephritis, multiple sclerosis, Crohn's disease, ulcerativecolitis, systemic lupus erythematosus and psoriasis.
 6. The combinationaccording to claim 1, comprising R,R-glycopyrrolate, formoterol and aglucocorticoid selected from budesonide, mometasone, and physiologicallyacceptable salts thereof.
 7. A pharmaceutical composition for thetreatment of inflammatory diseases comprising at least three differentpharmaceutically active substances or physiologically acceptable saltsthereof selected from the group consisting of anticholinergics,glucocorticoids, and β₂-adrenoceptor agonists.
 8. The pharmaceuticalcomposition according to claim 7, wherein the anticholinergic isR,R-glycopyrrolate or a physiologically acceptable salt thereof.
 9. Thepharmaceutical composition according to claim 7, wherein the compositionis an inhalable aerosol, with or without propellant, an inhalable drypowder, an inhalable suspension or an inhalable solution.
 10. Thepharmaceutical composition according to claim 8, wherein the compositionis in an inhaler.
 11. The pharmaceutical composition according to claim8, wherein composition is in a fixed or free combination forsimultaneous, sequential or separate administration together withacceptable excipients, adjuncts, and additives in a pharmaceutical formsuitable for inhalative application.
 12. The pharmaceutical compositionaccording to claim 8, comprising R,R-glycopyrrolate, formoterol and aglucocorticoid selected from budesonide and mometasone, orphysiologically acceptable salts thereof.
 13. A method for treating aninflammatory disease or an auto immune disease comprising administeringa combination of at least three different pharmaceutically activesubstances or physiologically acceptable salts thereof selected from thegroup consisting of anticholinergics, glucocorticoids, andβ₂-adrenoceptor agonists, wherein the inflammatory disease is selectedfrom the group consisting of respiratory diseases, and rheumatism orauto-immune diseases.
 14. The method of claim 13, wherein therespiratory disease is asthma or COPD.
 15. The method of claim 13,wherein the auto-immune disease is selected from the group consisting ofrheumatoid arthritis, glomerulonephritis, multiple sclerosis, Crohn'sdisease, ulcerative colitis, systemic lupus erythematosus and psoriasis.